Methods and Compositions for Detecting and Isolating Phosphorylated Molecules Using Hydrated Metal Oxides

ABSTRACT

The invention provides methods for detecting and isolating phosphomolecules using phosphoaffinity materials that comprise a hydrated metal oxide. In an embodiment, a method for detecting a phosphomolecule in a sample involves (a) contacting a sample with a phosphoaffinity material comprising a hydrated metal oxide, under conditions wherein a phosphomolecule is capable of binding to the phosphoaffinity material to form a phosphomolecule-phosphoaffinity material complex, and (b) detecting formation of a phosphomolecule-phosphoaffinity material complex, thereby detecting a phosphomolecule in the sample. In another embodiment, a method for isolating a phosphomolecule from a sample involves (a) contacting a sample with a phosphoaffinity material comprising a hydrated metal oxide, under conditions wherein a phosphomolecule is capable of binding to the phosphoaffinity material to form a phosphomolecule-phosphoaffinity material complex, wherein the hydrated metal oxide comprises yttrium, and (b) separating the phosphomolecule-phosphoaffinity material complex from the sample, thereby isolating the phosphomolecule from the sample.

BACKGROUND

The present invention relates to detecting and isolating phosphorylatedmolecules using phosphoaffinity materials containing one or morehydrated metal oxides, such as yttrium oxide, yttrium aluminum garnetand titanium dioxide.

Cells of the body contain many types of molecules that vary in function,size, lifetime, and numerous other characteristics. Some of thesemolecules are unchanged during their lifetime within the body, whileother molecules become modified through chemical reactions. Themodifications can be indicative of particular cell states, includingnormal states as well as abnormal states caused by injury, infection anddisease.

Proteins, for example, are often chemically modified during theirlifetime in the body. A protein can be modified during and after itssynthesis, or both, and the modification can change the size and thestructure of the protein, which in turn can result in changing theprotein's function or behavior in the cell. An example of a modificationof a protein is the addition of a phosphate group (phosphorylation).

Reversible phosphorylation of threonine, serine, and tyrosine residueson proteins by enzymes called kinases (which add a phosphate) andphosphatases (which remove the phosphate) plays an important role inregulating many cell processes, such as growth and cell cycle control.Phosphorylation can occur sequentially from one protein to another,resulting in a series of activations called a “phosphorylation cascade,”which is a type of “signal transduction pathway.” Phosphorylationcascades are recognized as signaling networks that direct growth, death,and differentiation of cells—the critical signals for maintaining normalcells in the body. At any given moment in a cell, determination ofphosphorylation states of proteins can indicate a signal transductionstate, for example an “on” or “off” state of cell growth.

Many cellular processes are regulated by reversible phosphorylation ofproteins and upwards of 30% of the total complement of proteinsexpressed by human cells are likely to be phosphorylated at some pointduring their existence. Determination of protein phosphorylation stateis thus important for identifying protein kinase substrates, as well asrevealing the on/off state of signal transduction pathways. The on/offstate of signal transduction pathways can be important to understandingpathophysiological processes, such as cancer. To better understand suchsignal transduction pathways, efforts are underway within the researchcommunity to identify phosphorylated proteins of various cell typesunder different cellular conditions, such as normal and diseasedconditions. Determination of differences in phosphorylation that occurunder normal and diseased conditions can be used, for example, indevelopment of diagnostic and other medical tests.

Given the important role of phosphorylation in signal transductionpathways, analysis of phosphorylation events that occur within theentire complement of proteins expressed by cells (phosphoproteomeanalysis), is useful for understanding a range of cellular processes.Phosphoproteome analysis likely will reveal insight into complexbiological processes, such as differentiation, growth control andregulated cell death. Accordingly, phosphoproteome analysis is expectedto contribute to development of diagnostic and prognostic tests, improveaspects of clinical trials, and provide indications of drug safety andefficacy during drug development.

One challenge in the field of phosphoproteome analysis is developingaccurate methods for global evaluation of protein phosphorylationlevels. Global analysis of protein phosphorylation is an analyticalchallenge because signaling phosphoproteins are typically present in lowabundance within cells. Analytical methods that improve global analysisof protein phosphorylation can contribute to development of medicaltests, such as tests that can simultaneously test for multiplephosphoprotein biomarkers. This type of test is expected to be helpfulfor detecting diseases and conditions for which single diagnosticmarkers are unfeasible or unavailable.

Thus, the ability to detect and/or isolate phosphoproteins is useful forcellular research as well as medical test development, given the centralrole of phosphorylation in many disease processes. Improved approachesfor phosphomolecule isolation and detection would accelerate proteinphosphorylation global analysis and related general and biomedicalphosphomolecule research.

SUMMARY

The technology described herein relates to methods for isolating aphosphomolecule from a sample. In one aspect, method involves (a)contacting a sample with a phosphoaffinity material comprising ahydrated metal oxide, under conditions wherein a phosphomolecule iscapable of binding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, and (b) separating thephosphomolecule-phosphoaffinity material complex from the sample,thereby isolating the phosphomolecule from the sample. In anotheraspect, the method involves (a) contacting a sample with aphosphoaffinity material comprising a hydrated metal oxide and asupport, under conditions wherein a phosphomolecule is capable ofbinding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, and (b) eluting aphosphomolecule from the phosphomolecule-phosphoaffinity materialcomplex, thereby isolating the phosphomolecule from the sample. Thesemethods further can involve separating the phosphomolecule from thephosphomolecule-phosphoaffinity material complex.

In an embodiment, the invention provides a method for isolating aphosphomolecule from a sample. The method involves (a) contacting asample with a phosphoaffinity material comprising a hydrated metaloxide, under conditions wherein a phosphomolecule is capable of bindingto the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, wherein the hydratedmetal oxide comprises yttrium, and (b) separating thephosphomolecule-phosphoaffinity material complex from the sample,thereby isolating the phosphomolecule from the sample. In anotherembodiment, a method for isolating a phosphomolecule from a sampleinvolves (a) contacting a sample with a phosphoaffinity materialcomprising a hydrated metal oxide and a support, under conditionswherein a phosphomolecule is capable of binding to the phosphoaffinitymaterial to form a phosphomolecule-phosphoaffinity material complex,wherein the hydrated metal oxide comprises yttrium, and (b) eluting aphosphomolecule from the phosphomolecule-phosphoaffinity materialcomplex, thereby isolating the phosphomolecule from the sample. Ifdesired, unbound sample components can be removed from thephosphomolecule-phosphoaffinity material complex prior to eluting. In anembodiment of these methods, the hydrated metal oxide is yttrium oxide.In another embodiment of these methods, the hydrated metal oxide isyttrium iron garnet.

The invention provides a method for isolating a phosphorylatedpolypeptide from a sample. The method involves (a) contacting a samplewith a phosphoaffinity material comprising a hydrated metal oxide and asupport, under conditions wherein a phosphomolecule is capable ofbinding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex and in a liquid mediumcomprising an organic solvent, and (b) eluting a phosphomolecule fromthe phosphomolecule-phosphoaffinity material complex, thereby isolatingthe phosphomolecule from the sample. In various embodiments, the liquidmedium can include, for example, isopropanol or acetonitrile. In anembodiment, the eluting is performed in the presence of a detergent,such as an ionic detergent.

The invention provides methods for detecting a phosphomolecule in asample. In an embodiment, the methods involve (a) contacting a samplewith a phosphoaffinity material comprising a hydrated metal oxide, underconditions wherein a phosphomolecule is capable of binding to thephosphoaffinity material to form a phosphomolecule-phosphoaffinitymaterial complex, and (b) detecting formation of aphosphomolecule-phosphoaffinity material complex, thereby detecting aphosphomolecule in the sample. In an embodiment, the detecting caninvolve measuring binding between the phosphomolecule andphosphoaffinity material. In another embodiment, the phosphoaffinitymaterial portion of the phosphomolecule-phosphoaffinity material complexis detected. In a specific embodiment, the metal of the phosphoaffinitymaterial portion is detected. In a further embodiment, thephosphomolecule portion of the phosphomolecule-phosphoaffinity materialcomplex.

The invention provides a variety of commercial packages useful forcarrying out a method for isolating and/or detecting a phosphomoleculein a sample. In an embodiment, a commercial package includes a hydratedmetal oxide attached to a support, wherein the hydrated metal oxidecomprises yttrium. In another embodiment, a commercial package includesa phosphoaffinity unit, the unit comprising a plurality of supportsheets coated with a hydrated metal oxide. The support sheet can be, forexample, a membrane or paper, such as cellulose. In a furtherembodiment, a commercial package contains a phosphoaffinity particlecomprising a hydrated metal oxide and a detectable agent that binds tothe hydrated metal oxide.

In any of the methods and commercial packages described herein, thehydrated metal oxide can be in, for example, particle form. As such, thephosphoaffinity material can comprise a support. The support can beselected from the group of particle, bead, gel, matrix, membrane,filter, fiber, sheet, mesh, frit, resin, sample vessel, column, pipettetip, slide channel and MALDI-TOF plate. The support can include adetectable tag, if desired.

The hydrated metal oxide used in a method of the invention can beselected from the group of aluminum oxide, titanium oxide, yttrium irongarnet, yttrium aluminum garnet, yttrium gallium garnet, ferric oxide,gallium oxide, yttrium oxide, vanadium oxide, zirconium oxide, irontitanate, iron aluminate, calcium titanate, sodium titanate, zirconiumaluminate, goethite, gibbsite, bayerite, boehmite, ilmenite,ilmenorutile, pseudorutile, rutile, brookite, pseudobrookite,geikielite, pyrophanite, ecandrewsite, melanostibite, armalcolite,srilankite and anatase. In particular, the metal oxide can be selectedfrom the group of yttrium oxide, yttrium iron garnet and titaniumdioxide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an exemplary process for isolating phosphorylated moleculesusing hydrated metal oxide particles in a multiwell microplatefiltration format.

FIG. 2 shows an exemplary process for isolating phosphorylated moleculesusing hydrated metal oxide/cellulose composite membranes in a multiwellmicroplate filtration format.

FIG. 3 shows an exemplary process for isolating phosphorylated moleculesusing a hydrated metal oxide surface on wells in a multiwell microplatefiltration format.

FIG. 4 shows an exemplary process for isolating phosphorylated moleculesusing magnetic particles coated with a hydrated metal oxide in amultiwell microplate filtration format.

FIG. 5 shows selective isolation of phosphorylated proteins using anyttrium oxide phosphoaffinity material. FIG. 5A shows a protein profilecorresponding to phosphopeptide-containing starting material; FIG. 5Bshows a protein profile corresponding to isolated phosphorylatedpeptide.

FIG. 6 shows selective isolation of phosphorylated peptides using anyttrium iron garnet oxide phosphoaffinity material. FIG. 6A shows aprotein profile corresponding to a phosphopeptide-containing startingmaterial; FIG. 6B shows a protein profile corresponding to isolatedphosphorylated peptide.

FIG. 7 shows selective isolation of phosphorylated peptides using atitanium dioxide phosphoaffinity material. FIG. 7A shows a proteinprofile corresponding to a phosphopeptide-containing starting material;FIG. 7B shows a protein profile corresponding to isolated phosphorylatedpeptide.

FIG. 8 shows positive control MALDI-TOF mass spectra of starting samplecontaining p60 c-src peptide (top) and phosphorylated p60 c-src peptide(pp60 c-src peptide) (bottom) spotted directly onto MALDIChip plates.

FIG. 9 shows MALDI-TOF mass spectra of a non-phosphorylated peptidefraction that did not bind to titanium dioxide particles (flow-throughfraction).

FIG. 10 shows MALDI-TOF mass spectra of a phosphorylated peptidefraction eluted from titanium dioxide particles.

FIG. 11 shows selective isolation of phosphorylated peptide from asample containing a phosphorylated peptide and a non-phosphorylatedpeptide, using a titanium dioxide phosphoaffinity membrane. FIG. 11Ashows a mass spectrum of starting material phosphorylated (P2P) andnon-phosphorylated (P2) peptides; FIG. 11B shows a mass spectrum ofpeptides that bound to titanium oxide coated membrane; FIG. 11C shows amass spectrum of peptides that did not appreciably bind to the membrane(flow-through fraction).

FIG. 12 shows selective isolation of phosphorylated peptides from aserum-containing sample using a titanium dioxide phosphoaffinitymembrane. FIG. 12A shows mass spectra of serum and serum spiked withphosphorylated (P2P) and non-phosphorylated (P2) peptides; FIG. 12Bshows mass spectra of peptides that bound to titanium oxide coatedmembrane; FIG. 12C shows mass spectra of peptides that did notappreciably bind to the membrane (flow-through fraction).

FIG. 13 shows selective isolation of phosphopeptides fromtrypsin-digested beta casein using a titanium dioxide phosphoaffinitymaterial. FIG. 13A shows mass spectra of peptides eluted from thephosphoaffinity material, peptides that did not bind (flow-through), andstarting material; FIG. 13B shows a mass spectrum corresponding tomultiple forms of phosphorylated peptides isolated and detected.

FIG. 14 shows selective isolation of phosphorylated ovalbumin from afive-protein mixture using a titanium dioxide phosphoaffinity material.FIG. 14A shows an image of an SDS-PAGE gel of eluted and flow-throughfractions; FIG. 14B shows normalized phosphoprotein enrichment ratios.

DESCRIPTION

The technology described herein relates to methods, compositions andcommercial packages for isolating and/or detecting phosphorylatedmolecules using hydrated metal oxide-containing phosphoaffinitymaterials.

In an embodiment, the present invention is directed to methods forisolating a phosphomolecule. The isolation methods are applicable topreparing populations of different types of phosphomolecules, as well asto preparing a single type phosphomolecule sample. The isolation methodscan be used for preparing samples enriched with phosphomolecules, forexample to improve detection of phosphomolecules in a complex sample.Isolation of phosphomolecules from a sample can be achieved by bindingthe phosphomolecules to the phosphoaffinity material and separating thephosphomolecule-phosphoaffinity material complex from the sample.Isolation also can be achieved by binding the phosphomolecule to thephosphoaffinity material, washing away unbound sample components, andeluting phosphomolecules from the phosphoaffinity material. Use of themethods of the invention for preparing isolated phosphorylatedpolypeptides is described herein, for example, in Examples 1, 2 and 3.

A phosphoaffinity material used in the methods and commercial packagesdescribed herein contain, incorporate or are solid forms of a hydratedmetal oxide, and the hydrated metal oxide binds selectively to thephosphomolecules. As is described below, a variety of hydrated metaloxides are suitable for binding to phosphomolecules. Example 1 describesindividual use of three different phosphoaffinity materials—yttriumoxide, yttrium iron garnet and titanium oxide—for selectively binding tophosphoproteins. Once a complex of the phosphoaffinity material andphosphomolecule is formed, the complex itself can be separated from thesample and/or the phosphomolecule portion of the complex can beseparated. As is described herein below, such separations can be carriedout using a variety of means, depending on the format selected by theuser. For example, when the phosphoaffinity material is in solidparticle form or is incorporated into a support, separation can beperformed by separating the particle or support (solid or semi-solidphase) from the sample (liquid phase), or visa versa.

The isolation methods described herein can be performed in a variety ofphysical formats. For example, phosphomolecules can be eluted fromcolumn-packed phosphoaffinity materials (see Example 1);phosphomolecules can be eluted from particles packed into wells of amultisample plate (see Example 2); phosphomolecule samples prepared asdescribed in Examples 1 and 2 can be subjected to further purificationprior to analysis (see Example 3); and phosphomolecules can be elutedfrom membranes coated with a phosphoaffinity material (see Examples 5, 7and 8). Given the examples and guidance provided herein, it will berecognized that a phosphoaffinity material incorporating a hydratedmetal oxide can be used in macro, micro, low-throughput andhigh-throughput formats.

As used herein, the term “isolating” when used in reference to aphosphomolecule means the act of separating the phosphomolecule fromother molecules, substances or materials in the sample. The term“isolated” when used in reference to a phosphomolecule, phosphoaffinitymaterial, metal oxide or other component useful in a method orcommercial package of the invention means that the component is actedupon by the hand of man to remove other molecules, substances ormaterials with which the component is associated in a sample orpreparation. The term isolated does not require absolute purity, butrather is intended as a relative term. As such, the term isolatingincludes acting on a sample to increase the amount of phosphomoleculesin the sample relative to the amount of one or more initial samplecomponents or amount of initial phosphomolecules, which is sometimesreferred to herein as enriching a sample.

A method of the invention can be used for isolating or enriching aphosphomolecule from samples of varying complexity. Examples 1, 2, 5 and8 describe enrichment of phosphopeptides in samples containing a peptidein phosphorylated state and a non-phosphorylated state; Example 6describes enrichment of phosphoproteins in a sample containing humanserum; Example 7 describes enrichment of phosphopeptides in a samplecontaining trypsin-digested protein; and Example 8 describes enrichmentof phosphoproteins in a sample containing a 5-protein mixture.

As is described in Example 8, the amount of a phosphomolecule in asample can be enriched by binding the sample to a phosphoaffinitymaterial containing a hydrated metal oxide; removing unbound samplecomponents from the phosphoaffinity material; and elutingphosphomolecules from the phosphoaffinity material. In this specificexample, titanium dioxide coated membranes were used as thephosphoaffinity material and phosphoprotein enrichment was determined bycomparing the amount of phosphoprotein before and after performing theisolation, relative to the amount of control proteins bovine serumalbumin, carbonic anhydrase and myoglobin before and after performingthe isolation. Ovalbumin enrichments of 9.5, 8 and 11 relative to BSA,CAH and myo, respectively, were observed in the absence of detergent,while enrichments of 17.7, 8.4 and 125.7, respectively, were observed inthe presence of detergent.

In an embodiment, the present invention is directed to methods fordetecting a phosphomolecule. Detection of a phosphomolecule in a samplecan be achieved by binding the phosphomolecule to a phosphoaffinitymaterial and detecting the complex of the phosphoaffinity material andphosphomolecule, or a portion of the complex. As is described hereinbelow, numerous and diverse analytical methods can be applied todetecting such a complex or a portion of the complex. For example, aphysiochemical property of a complex relative to its components, such asmass, charge to mass ratio, refractive index, fluorescence anisotropyand the like can be detected. As another example, a property resultingfrom the proximity between phosphomolecule and phosphoaffinity materialwhen complexed, such as fluorescence resonance energy transfer andradiometric scintillation proximity-based emission, can be detected. Asa further example, a component of the complex, such as thephosphomolecule or the hydrated metal oxide can be detected. Suchdetecting can involve directly detecting the phosphomolecule or hydratedmetal oxide or detecting a tag on the phosphomolecule or hydrated metaloxide. Detection of the metal portion of phosphomolecule-phosphoaffinitymaterial complex is described in Examples 11 and 12.

The detection methods described herein can be performed in a variety ofphysical formats. For example, phosphomolecules can be detected when insolution; when in a matrix (see Example 10); when in an array (seeExample 9); as well as other formats. A variety of particle-basedmethods for detecting a phosphomolecule are described herein (see, forexample, Examples 10 and 11). A phosphoaffinity particle, which can befor example, a hydrated metal oxide or a particle support coated withhydrated metal oxide, can be detected directly; can be labeled prior todetection; or can be used to enrich or isolate aphosphomolecule-phosphoaffinity material complex which is then detected.

The technology described herein involves formation of a complex betweena phosphomolecule and a phosphoaffinity material made of, or containing,a hydrated metal oxide. Previous studies have shown that particularphosphomolecules can bind to certain transition metal cations. Inorganicorthohosphates have been shown to adsorb to titanium dioxide particles(Damen et al, 1991; Kim and Chung, 2001). Organic phosphates such asphosphorylated peptides have been shown to bind selectively to titaniumdioxide particles, and titanium dioxide particles have been used forenriching phosphoproteins in HPLC techniques (Sano and Nakamura, 2004a,b). Phosphorylated organic compounds, including pyridoxal 5′phosphate-containing and phosphomannose-containing proteins, have beenshown to bind selectively to aluminum oxide particles (Pugniere et al.,1988; Coletti-Previero and Previero, 1989; Koppel et al, 1994). Iron(III) oxyhydroxide particles have been shown to adsorbphosphate-containing compounds and have been used in treatment ofindustrial waste water (Zeng et al, 2004; Mustafa et al, 2004).Monoesters of phosphoric acid and phosphonic acids have been shown tobind to hydrated metal oxides over a wide pH range.D,L-serine-O-phosphate, ethanolamine-O-phosphate and phenylphosphonicacid have been shown to bind selectively to hydrated aluminum oxide(Coletti-Previero and Previero, 1989) and adenosine 5′-phosphate hasbeen shown to bind selectively to aluminum oxide (Colefti-Previero andPreviero, 1989). Immobilized metal affinity chromatography (IMAC) uses astationary phase containing organic chelating groups charged withtrivalent transition metal ions, such as Ga (III) and Fe(III), to enrichphosphopeptides prior to microchemical analysis (Posewitz and Tempst,1999). For IMAC, peptides are eluted from the resin using a bufferhaving higher pH or higher concentration of inorganic phosphate withrespect to the sample loading buffer.

An example of a commercial IMAC-based procedure is the IMAP assay(Molecular Devices, Sunnyvale, Calif.). IMAP is a fluorescencepolarization homogenous solution assay in which beads derivatized withtrivalent transition metal ions are used for binding to phosphateresidues. The beads are added to a kinase reaction along with afluorescently-labeled peptide substrate. If the kinase phosphorylatesthe substrate, the bead binds to the phosphate residue. Rotation of thefluorescent phosphorylated substrate is slowed by the bead binding,resulting in greater polarization of the emitted light. IMAP appears tobe applicable to measurement of phosphopeptides but not phosphoproteins.In IMAP, fluorescence polarization readings are performed at a pH valueof less than about 6.0 to preserve interaction of the phosphate groupwith the trivalent cation. Consequently, continuous monitoring of kinaseassays cannot be achieved by IMAP because kinase reactions are typicallyinhibited at the low pH at which fluorescence polarization is read.

The technology described herein for isolating and/or detectingphosphomolecules differs from isolation methods using IMAC, in that thepresent technology employs a hydrated metal oxide, rather than achelated trivalent transition metal cation. The hydrated metal oxideused in the present technology can be presented to a sample in variousforms, including particles and films. Such particles and films differfrom metal ions in their physical properties, such as theircharacteristic surface plasmon absorption bands. As another example,whereas metal ions do not have isoelectric points, hydrated metal oxideparticles and surfaces have isoelectric points. The mechanism ofphosphate moiety-metal oxide interactions on surfaces is thought toinvolve physiochemical phenomenon different from interaction withtransition metal ions. Without wishing to be bound by theory, it appearsthat interaction of phosphate moieties with hydrated metal oxide occursvia an ion exchange sorption-type mechanism in which exchange ofhydroxide anions on the surface of the metal oxide with phosphatemoieties mediate the sorption. For this reason, experimental conditionsfor hydrated metal oxide interaction with phosphate moieties can differfrom immobilized metal ion interaction with phosphate moieties. Inparticular, hydrated metal oxide phosphoaffinity materials do notinvolve loading metal ions onto an affinity material and do not leachmetal ions during use. In addition, devices made from hydrated metaloxides can be rugged and autoclavable.

The technology described herein relates to methods for isolating aphosphomolecule from a sample. In one aspect, method involves (a)contacting a sample with a phosphoaffinity material comprising ahydrated metal oxide, under conditions wherein a phosphomolecule iscapable of binding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, and (b) separating thephosphomolecule-phosphoaffinity material complex from the sample,thereby isolating the phosphomolecule from the sample. In anotheraspect, the method involves (a) contacting a sample with aphosphoaffinity material comprising a hydrated metal oxide and asupport, under conditions wherein a phosphomolecule is capable ofbinding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, and (b) eluting aphosphomolecule from the phosphomolecule-phosphoaffinity materialcomplex, thereby isolating the phosphomolecule from the sample. Thesemethods further can involve separating the phosphomolecule from thephosphomolecule-phosphoaffinity material complex.

In an embodiment, the invention provides a method for isolating aphosphomolecule from a sample. The method involves (a) contacting asample with a phosphoaffinity material comprising a hydrated metaloxide, under conditions wherein a phosphomolecule is capable of bindingto the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, wherein the hydratedmetal oxide comprises yttrium, and (b) separating thephosphomolecule-phosphoaffinity material complex from the sample,thereby isolating the phosphomolecule from the sample. In anotherembodiment, a method for isolating a phosphomolecule from a sampleinvolves (a) contacting a sample with a phosphoaffinity materialcomprising a hydrated metal oxide and a support, under conditionswherein a phosphomolecule is capable of binding to the phosphoaffinitymaterial to form a phosphomolecule-phosphoaffinity material complex,wherein the hydrated metal oxide comprises yttrium, and (b) eluting aphosphomolecule from the phosphomolecule-phosphoaffinity materialcomplex, thereby isolating the phosphomolecule from the sample. Ifdesired, unbound sample components can be removed from thephosphomolecule-phosphoaffinity material complex prior to eluting. In anembodiment of these methods, the hydrated metal oxide is yttrium oxide.In another embodiment of these methods, the hydrated metal oxide isyttrium iron garnet.

The invention provides a method for isolating a phosphorylatedpolypeptide from a sample. The method involves (a) contacting a samplewith a phosphoaffinity material comprising a hydrated metal oxide and asupport, under conditions wherein a phosphomolecule is capable ofbinding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex and in a liquid mediumcomprising an organic solvent, and (b) eluting a phosphomolecule fromthe phosphomolecule-phosphoaffinity material complex, thereby isolatingthe phosphomolecule from the sample. In various embodiments, the liquidmedium can include, for example, isopropanol or acetonitrile. In anembodiment, the eluting is performed in the presence of a detergent,such as an ionic detergent.

The invention provides methods for detecting a phosphomolecule in asample. In an embodiment, the methods involve (a) contacting a samplewith a phosphoaffinity material comprising a hydrated metal oxide, underconditions wherein a phosphomolecule is capable of binding to thephosphoaffinity material to form a phosphomolecule-phosphoaffinitymaterial complex, and (b) detecting formation of aphosphomolecule-phosphoaffinity material complex, thereby detecting aphosphomolecule in the sample. In an embodiment, the detecting caninvolve measuring binding between the phosphomolecule andphosphoaffinity material. In another embodiment, the phosphoaffinitymaterial portion of the phosphomolecule-phosphoaffinity material complexis detected. In a specific embodiment, the metal of the phosphoaffinitymaterial portion is detected. In a further embodiment, thephosphomolecule portion of the phosphomolecule-phosphoaffinity materialcomplex.

A phosphomolecule can be isolated and/or detected from a variety oftypes of samples using the technology described herein. As used herein,the term “sample” means a substance that contains or is suspected ofcontaining a phosphorylated molecule. A sample useful in a method of theinvention for isolating and/or detecting a phosphorylated molecule canbe a liquid or solid, can be dissolved or suspended in a liquid, can bein an emulsion or gel, and can be bound to or absorbed onto a material.A sample can be a biological sample, environmental sample, experimentalsample, diagnostic sample, or any other type of sample that contains oris suspected to contain a phosphorylated molecule. As such, a sample canbe, or can contain, an organism, organ, tissue, cell, bodily fluid,biopsy sample, or fraction thereof. A sample useful in a method of theinvention can be any material that is suspected to containphosphorylated molecules, such as substrates of kinases andphosphatases. In a biological context, a sample can include biologicalfluids, whole organisms, organs, tissues, cells, microorganisms, culturesupernatants, subcellular organelles, protein complexes, individualproteins, recombinant proteins, fusion proteins, viruses, viralparticles, peptides and amino acids.

A sample can be processed to preserve or stabilize phosphorylatedmolecules. Methods for preserving the integrity of molecules in a sampleare well known to those skilled in the art. Such methods include the useof appropriate buffers and/or inhibitors, including nuclease, proteaseand phosphatase inhibitors that preserve or minimize changes in themolecules in the sample. Such inhibitors include, for example, chelatorssuch as ethylenediamne tetraacetic acid (EDTA), ethylene glycolbis(P-aminoethyl ether)N,N,N1,NI-tetraacetic acid (EGTA), proteaseinhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin,leupeptin, antipain and the like, and phosphatase inhibitors such asphosphate, sodium fluoride, vanadate and the like. Appropriate buffersand conditions for allowing selective interactions between molecules arewell known to those skilled in the art and can be varied depending, forexample, on the type of molecule in the sample to be characterized (see,for example, Ausubel et al., Current Protocols in Molecular Biology(Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane,Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999);Tietz Textbook of Clinical Chemistry, 3rd ed., Burtis and Ashwood, eds.,W.B. Saunders, Philadelphia, (1999)).

A sample also can be processed to reduce the presence of interferingsubstances and/or reduce non-selective binding of sample components to aphosphoaffinity material. Exemplary agents useful for improvingsolubility of phosphorylated molecules include detergents such as TRITONX-100, sodium deoxycholate, urea, thiourea and sodium dodecyl sulfate. Atendency of acidic polypeptides to bind to phosphoaffinity materialsnon-selectively can be reduced by methyl esterification of thepolypeptide sample (Ficarro et al, 2002; Brill et al, 2004).

A sample can be fractionated prior to use in a method of the inventionif desired. Well known fractionation methods such asimmunoprecipitation, 1-D gel electrophoresis, 2-D gel electrophoresis,electroblotting, liquid chromatography, electrochromatography, dialysis,two-phase polymer separations and solid phase extraction can be used forsample fractionation. Various methods for fractionating a fluid sampleor cell extract are well known to those skilled in the art, includingsubcellular fractionation or chromatographic techniques such as ionexchange, hydrophobic and reverse phase, size exclusion, affinity,hydrophobic charge-induction chromatography, and the like (Ausubel etal., supra, 1999; Scopes, Protein Purification: Principles and Practice,third edition, Springer-Verlag, New York (1993); Burton and Harding, J.Chromatoqr. A 814:71-81 (1998)).

A sample can be labeled with a tag prior to use in a method of theinvention. Examples of tags include detectable moieties, such aluminescent moieties, fluorescent moieties, radioactive moieties and thelike; purification tags such as polyhistidine, flag, myc and GST tags;polynucleotide tags, aptamers, protein nucleic acids; biological tagssuch as phage; antibody and antibody-like tags; reactive organicmolecule or peptide mass tags or other mass tags such as particles ofdefined size, for example, metal beads and nanoparticle tags, and thelike. Example 11 describes labeling of a sample with a fluorescentmoiety. In this example, detection of a phosphomolecule is performed bydetecting fluorescence quenching of the phosphomolecule tag.

A phosphoaffinity material useful in a method of the invention forisolating and/or detecting a phosphomolecule or commercial packagecontains or is a hydrated metal oxide that binds selectively to aphosphomolecule. Exemplary hydrated metal oxides include hydrated formsof yttrium oxide (Y₂O₃), iron oxide (Fe₂O₃, Fe₃O₄), yttrium iron garnet(Fe₅O1₂Y₃), yttrium gallium garnet (Y₃Ga₅O1₂), yttrium aluminum garnet(Y₃Al₅O₁₂), vanadium oxide (VO₂), zirconium oxide (ZrO₂), iron titanate(Fe₂Ti₂O7), iron aluminate (FeAl₂O₅), calcium titanate (CaTiO₃), sodiumtitanate (Na₂TiO₃), and zirconium titanium aluminate (ZrTi₂Al₂O₅).Hydrated metal oxides also include composites of cores of hematite,magnetite, chromium hydroxide or titanium dioxide, with surface coatingssuch as alumina, titanium dioxide, zirconia and yttria. Mineral oxidesare widely distributed in nature and are often present as suspendedparticles in aqueous environments. Chemical processes, such as sorptionphenomena, are determined in part by surface properties of the particlesat the metal oxide-water interface. A number of naturally occurringmineral oxides, such as goethite (α-FeOOH), gibbsite (α-Al(OH)₃),bayerite (β-Al(OH)₃), boehmite (γ-Al(OH)₃), ilmenite (FeTiO₃),ilmenorutile (Fex(Nb, Ta)₂x.₄Ti1-xO₂), pseudorutile (Fe₂Ti₃O₉), rutile(TiO₂), brookite (TiO₂), pseudobrookite (Fe₂TiO₅), geikielite (MgTiO₃),pyrophanite (MnTiO₃), ecandrewsite (Zn, Fe, Mn)TiO₃, melanostibite (Mn(Sb, Fe)O₃), armalcolite (Mg, Fe)Ti₂O5, srilankite (Ti, Zr)O₂ andanatase (TiO₂), can be used in a phosphoaffinity material for selectivebinding to phosphomolecules. In general, these and other inorganicmetals, when hydrated, present a surface that is covered with a layer ofa metal oxide, hydroxide or oxohydroxidehydroxyl groups which contributeto their overall physicochemical properties, including their ability toadsorb phosphorylated molecules. In an embodiment, a phosphoaffinitymaterial useful in the methods and commercial packages of the inventioncontain a hydrated metal oxide selected from the group of aluminumoxide, titanium oxide, yttrium iron garnet, yttrium aluminum garnet,yttrium gallium garnet, ferric oxide, gallium oxide, yttrium oxide,vanadium oxide, zirconium oxide, iron titanate, iron aluminate, calciumtitanate, sodium titanate, zirconium titanium aluminate, goethite,gibbsite, bayerite, boehmite, ilmenite, ilmenorutile, pseudorutile,rutile, brookite, pseudobrookite, geikielite, pyrophanite, ecandrewsite,melanostibite, armalcolite, srilankite and anatase. In specificembodiments, the hydrated metal oxides are yttrium oxide, yttrium irongarnet and titanium dioxide (see, for example, Example 1). In aparticular embodiment, the selected hydrated metal oxide is not titaniumdioxide, such as a non-titanium hydrated metal oxide. In otherembodiments, the selected hydrated metal oxide is not iron oxide and notaluminum oxide.

A phosphoaffinity material selected for use in a method or commercialpackage of the invention for isolating and/or detecting aphosphomolecule is capable of binding to a phosphomolecule. Aphosphomolecule can be a macromolecule, such as a polypeptide andpolynucleotide, as well as a small molecule, such as an amino acid andnucleotide. Non-limiting examples of molecules that can contain aphosphorylated moiety include an amino acid, a peptide, a polypeptide, anucleotide, a polynucleotide, a lipid, glycan and a carbohydrate. Aphosphorylated moiety present on a phosphorylated polypeptide, such as aprotein or peptide, can be phosphoserine, phosphothreonine,phosphotyrosine, 1-phosphohistidine, 3-phosphohistidine, phosphoasparticacid, phosphoglutamic acid, Nε-phospholysine,delta-O-phosphohydroxylysine, Nω-phosphoarginine, thiophosphorylation,phosphocysteine, pyridoxal phosphate Schiff base conjugated to theε-amino group of lysine, N-acetylglucosamine 1-phosphate modifiedserine, mannose 6-phosphate present in asparagine-linkedoligosaccharides or O-pantetheine phosphorylated serine.Phosphomolecules isolated and/or detected using a method of theinvention include molecules containing one or more phosphomimeticgroups. Non-limiting examples of phosphomimetic groups includeO-boranophosphopeptides and O-dithiophosphopeptides, derivatized ontyrosine, serine, or threonine residues, phosphoramide, H-phosphonate,alkylphosphonate, phosphorothiolate, phosphodithiolate andphosphorofluoridate. Selective binding means that the phosphoaffinitymaterial binds to one or more phosphomolecules but does notsubstantially bind to non-phosphomolecules.

It is understood that a particular phosphoaffinity material used in amethod or commercial package of the invention can be capable ofselective binding to phosphomolecules, or to a subset of these types ofmolecules, such as selective binding to phosphorylated polypeptides, aswell as to particular phosphorylated moieties. As an example ofselective detection of a particular phosphorylated moiety, detection ofphosphotyrosine residues on a phosphorylated polypeptide is described inExample 9.

A sample or phosphoaffinity material used in a method or commercialpackage of the invention can be attached to a support. As used herein,the term “support” means a solid or semi-solid material onto which ametal oxide, sample or phosphomolecule can be deposited, attachedimmobilized, entrapped, captured or coated, or which can befunctionalized to include a metal oxide, sample or phosphomolecule. Asupport can be a natural or synthetic material, and can be an organic orinorganic material, such as a polymer, resin, metal or glass. Suitablesupports are known in the art and illustratively include an agarose,such as is commercially available as Sepharose; a cellulose,illustratively including a carboxymethyl cellulose; a dextran, such asis commercially available as Sephadex; a polyacrylamide; a polystyrene;a polyethylene glycol; a resin; a silicate; divinylbenzene;methacrylate; polymethacrylate; glass; ceramics; paper; metals;metalloids; polyacryloylmorpholide; polyamide;poly(tetrafluoroethylene); polyethylene; polypropylene;poly(4-methylbutene); poly(ethylene terephthalate); rayon; nylon;poly(vinyl butyrate); polyvinylidene difluoride (PVDF); silicones;polyformaldehyde; cellulose acetate; cotton; wool; dextran; Trisacryl;hydroxyalkyl methacrylate, poly(vinylacetate-co-ethylene), oxiraneacrylate, polyethylene, polypropylene, poly(vinyl chloride), poly(methylmethacrylate), phenol resin, poly (vinylidene difluoride), poly(ethyleneterephthalate), polyvinylpyrrolidone, polycarbonate, starch,nitrocellulose; mixtures thereof, and the like.

A support useful in a method of the invention can have a variety ofphysical formats, which can include for example, a membrane, column, ahollow, solid, semi-solid, pore or cavity containing particle such as abead, a gel, a fiber, including a fiber optic material, a sheet, amatrix and sample receptacle. Non-limiting examples of samplereceptacles include sample wells, tubes, capillaries, vials and anyother vessel, groove or indentation capable of holding a sample,including those containing membranes, filters, matrices and the like. Asample receptacle can be contained on a multi-sample platform, such as amicroplate, slide, microfluidics device, array substrate, massspectrometry sample plate, and the like. A particle to which aphosphoaffinity material is attached can have a variety of sizes,including particles that remain suspended in a solution of desiredviscosity, as well as particles that readily precipitate in a solutionof desired viscosity. In particular embodiments, a particle support orphosphoaffinity material particle such as a crystal have diameters ofbetween about 1 nm and 1 μm. The term “phosphoaffinity particle” means aphosphoaffinity material in particle form. The term encompassesparticles coated with a phosphoaffinity material as well as particlesmade of a phosphoaffinity material, such as a crystal or other solidform. The term “phosphoaffinity sheet” means a phosphoaffinity materialin flat form, such as a paper, membrane, filter, and the like. Aphosphoaffinity material can be part of or incorporated into a device,such as for example, a spin-column, microcolumn pipette tip, multi-wellmicrowell strip, multi-well microplate and magnetic separator. A supportcan also contain a ferromagnetic or paramagnetic substance, for example,when magnetic separation procedures are employed.

If desired, a support can include a tag, such as a tag useful fordetection and/or purification. A support also can be an inherentcharacteristic of a hydrated metal oxide, such as a metal oxideparticle, crystal or other solid form. For use as a phosphoaffinitymaterial in column, bed or surface form, the support can havecharacteristics such as uniform porous network and chemical and/orbiological inertness.

A variety of procedures can be used for attaching or depositing a metaloxide onto a support for preparing a phosphoaffinity material useful ina method or commercial package of the invention. For example, the metaloxide can be deposited on the support through liquid-phase deposition,chemical bath deposition, successive ion layer adsorption and reaction(SILAR), electroless deposition, reactive sputtering, reactiveevaporation, spray pyrolysis, track-etching, anodic oxidation,cold-press molding, chemical vapor deposition, or sol-gel processing.The deposited metal oxide can be crystalline, nanocrystalline, poorlycrystallized or amorphous. In some embodiments, a crystalline layer issubsequently hydroxylated to render it suitable for bindingphosphorylated molecules, and the crystalline layer can be hydroxylatedby incubation in an aqueous-based medium for a period of time, such as,for example, one hour to several months.

In an embodiment, a metal oxide is attached to a support at aboutambient temperature and in an aqueous-based medium. In this embodiment,an organic support material is generally employed. Non-limiting examplesof organic support materials include cellulose, cotton, wool, dextran,agarose, polyacrylamide, Trisacryl, hydroxyalkyl methacrylate,poly(vinylacetate-co-ethylene), oxirane acrylate, polyethylene,polypropylene, poly(vinyl chloride), poly(methyl methacrylate), phenolresin, poly (vinylidene difluoride), poly(ethylene terephthalate),polyvinylpyrrolidone, polycarbonate and starch. Deposition can beachieved on an ion-by-ion or particle attachment basis.Functionalization of the organic support, such as with sulfonate,hydroxyl or carboxyl groups can aid in depositing the metal oxide. Inone embodiment, the hydrated metal oxide is deposited on to a support ofcellulose or modified cellulose. Therefore, in an embodiment, an organicsupport used in a method or commercial package of the invention isfunctionalized with organic groups, while in other embodiments, theorganic support is functionalized with sulfonate, hydroxyl or carboxylgroups.

In an embodiment, a hydrated metal oxide is deposited or attached to aninorganic support. Exemplary incorganic supports include ceramic, metal,glass, alumina, silica, zirconia, a ferromagnetic material and aparamagnetic material. More durable porous ceramic-based supports, suchas alumina, permit derivatization with hydrated metal oxides usingharsher conditions. Ceramic membranes can be useful for certainbiomedical applications because they are generally inert towards variousharsh chemicals (strong acids and organic solvents) and hightemperatures. For example, hydrated metal oxides are known to adsorbsilicate species from solution when they are stored in glass at neutralto basic pH values, resulting in a substantial decrease in isoelectricpoint of the material. One approach to removing adsorbed material fromhydrated metal oxides is to wash them with 1 M sodium hydroxide,deionized water, 1 M nitric acid and then again with deionized water.Whether an organic or inorganic support is employed, the hydrated metaloxide binds to the phosphomolecule with sufficient affinity to allowdetection of the complex, and is stable over the time of assayperformance.

One approach to preparing a porous hydrated metal oxide involveslow-temperature synthesis of thin films through direct deposition fromaqueous-based solutions using Ti (IV), Fe (III), Zr (IV) or Al (III)ions as precursors for the formation of hydrated metal oxide coatings(Niesen and De Guire, 2001). These metal ions can be used for thisgeneral approach because they are readily hydrolyzed, even in acidicaqueous-based media. Similar coatings can be prepared using alternativesolvents, such as 2-propanol or appropriately formulated blends ofacetic acid, acetone and water, without damaging the underlying organicsupport. Aqueous-based solutions of 0.03 to 0.1 M titanium tetrafluoridein the pH range of 1.0 to 3.1 at 40 to 70 degrees centigrade can be usedfor deposition of anatase films on both organic and inorganicsubstrates. Particularly, 0.05 M titanium tetrafluoride at pH 1.9 can beused for coating organic supports at 60 degrees centigrade.

Without wishing to be bound by theory, it appears that heterogeneousnucleation of titanium dioxide is required for effective coating ofsupports. Since titanium-fluoride bonds are relatively stable,hydrolysis and polymerization of the reactant occurs at a relativelyslow rate. At pH values above 3.1, homogenous nucleation ofsupersaturated solutions predominates and precipitation results. BelowpH values of 1.0, titanium tetrafluoride appears to form a metastablesolution with a slow reaction rate. Heterogeneous nucleation occurs atintermediate conditions between the metastable and supersaturatedstates. A phase diagram of the metal oxide starting materialconcentration versus pH for a given temperature can be used fordetermining appropriate reaction conditions for a variety of suitablestarting materials, including monomeric titanium tetrafluoride, titaniumlactate, titanium tetraisopropoxide, and titanium tetrabutoxide.

Once the reaction conditions for heterogeneous nucleation using theabove-described or different metal oxide starting materials aredetermined, the support can be incubated in the appropriate depositionsolution, generally for a period of 0.5 to 260 hours, depending on thedesired coating thickness, particular metal oxide and material to becoated. The diameter of the particles in the films typically increasesgradually throughout the deposition time course. Incubation time can beadjusted to achieve a desired layer thicknesses, such as a thickness of200 nm or less. Particle sizes in the films typically range from a fewnanometers to a few tens of nanometers.

Optionally, a surfactant can be included in the deposition solution toreduce any cracking or crazing resulting from bending stress applied tothe organic support subsequent to manufacture of the membranes.Non-limiting examples of surfactants that can be employed for thispurpose, as well as for other purposes employing detergents as describedthroughout the present application, include sodium dodecyl sulfate,lithium dodecyl sulfate, sodium bis-2-ethylhexylsulphosuccinate, sodiumcholate, perfluordecyl bromide, cetyltrimethylammonium bromide,didodecylamonium bromide, Triton X-100, polyoxyethylene 10-oleyl ether,polyoxyethylene-10-dodecyl ether, N,N-dimethyldodecylamine-N-oxide, Brij35, Tween-20, Tween-80, sorbitan monooleate, lecithin,diacylphosphatidylcholine, sucrose monolaurate and sucrose dilaurate. Abinder can be included in an aqueous- or organic-based metal oxidedeposition solution. The binder can be, for example, polyvinyl alcohol,polyethylene glycol, polyethyleneimine, poly(dimethylsiloxane),hydroxypropylcellulose and polyacrylamide. For metal oxides, generallyhydrophilic supports can have more favorable coating performancecharacteristics than hydrophobic ones, because binding of metal oxideparticles appears to involve formation of hydrogen bounds or bridgingoxygen atoms through a dehydration reaction between the particles andthe underlying substrate.

To remove excess fluoride that can remain affixed to a hydrated metaloxide surface when using fluoride-containing starting materials indeposition solutions, an aqueous-based solution containing fluoridescavengers, such as boric acid, can be employed. In some embodiments,titanium tetrafluoride, titanium lactate, titanium tetraisopropoxideand/or titanium tetrabutoxide can serve as precursor metal ions fordeposition of hydrated metal oxides onto a support. Aqueous-basedsolutions containing 0.03 to 0.1 M titanium tetrafluoride in the pHrange of 1.0 to 3.1 and in the temperature range of 40 to 70° C. can beemployed for deposition of the hydrated metal oxides onto thescaffolding. In embodiments, an aqueous-based solution comprising 0.05 Mtitanium tetrafluoride at pH 1.9 and at 60° C. is employed fordeposition of the hydrated metal oxides onto the support.

In another liquid-phase deposition approach, hydrated metal oxide orhydroxide thin films can be formed by the ligand-exchange (hydrolysis)equilibrium reaction of metal-fluoro complex ionic species and afluoride consumption reaction using boric acid or aluminum metal (Niesenand De Guire, 2001). Using this technique, thin films can be formed on avariety of organic substrates by immersing the supports directly in thedeposition solution. Aqueous solutions of ammonium titanium fluoride((NH₄)₂TiF₆) and boric acid (H₃BO₃), for example, can be used forcoating organic supports with titanium dioxide using this approach.Similar coatings can be produced for vanadia, iron oxyhydroxide,zirconia and multicomponent films containing more than one hydratedmetal oxide (Niesen and De Guire, 2001).

In addition to liquid phase deposition, aqueous-based methods such aschemical bath deposition, successive ion layer adsorption and reaction(SILAR), and electroless deposition can be employed to coat supportswith hydrated metal oxides (Niesen and DeGuire, 2001). As anotheralternative to the liquid-phase deposition approaches described above,coating can be performed by mixing of metal oxide powder with a solutioncontaining an appropriate binder, such as polyvinyl alcohol,polyethylene glycol, polyethyleneimine, poly(dimethylsiloxane),hydroxypropylcellose or polyacrylamide, and then depositing the materialon a ceramic support as a thin film. Additionally, hydrated metal oxidecoatings can be prepared by a range of other deposition techniques,including reactive sputtering, reactive evaporation, spray pyrolysis,track-etching, anodic oxidation, cold-pressed molding, chemical vapordeposition and sol-gel processing. In general, these methods require aheating process at relatively high temperatures, above 400 degreescentigrade, to obtain sufficient crystallinity. Ceramic-based membranes,for example, can be fabricated using these and other approaches.

Crystalline hydrated metal oxide coatings have been described. Forexample, titanium dioxide thin films have been used as antibacterialcoatings, as deodorization disinfection sheets, for soil-proofinghousehold furnishings, as anti-algal oil-proofing plates, as antifoggingcoatings, as deodorant fibers, and as a low-cost light-harvestingcomposite material for solar cells (Niesen and De Guire, 2001).

Crystallinity of hydrated metal oxide surfaces can be a usefulcharacteristic for certain uses of metal oxide-coated membranesmentioned above. Hydrated metal oxides can be nano-porous includeultrafine crystallites or be poorly crystallized, as long as theaffinity surface contains numerous hydroxide groups. The act ofsintering the metal oxide surface at elevated temperatures, such as attemperatures greater than 300 degrees centigrade, is expected to bedetrimental to the envisioned application, as this will reduce theextent of interaction between the metal oxide and the phosphorylatedmolecules, due to a loss of pendant hydroxyl groups on the oxidesurface. Thus, when sintered metal oxide surfaces are employed, aprocedure for regenerating the hydroxylated surface is generallyemployed after the heating process. A procedure for recovering hydroxylgroups involves incubating the metal oxides in an aqueous environmentfor an extended period of time.

The methods described herein are carried out under conditions that allowa phosphomolecule to bind a phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex. A phosphomoleculegenerally will bind to a phosphoaffinity material under typical proteininteraction assay conditions. Such conditions are well known to thoseskilled in the art and generally include roughly physiologically saltlevels, a buffering agent, and a temperature in the range of 4-37degrees C. For a chosen phosphoaffinity material, a sample can beadjusted or placed into a solution or environment to have a specifiedcharacteristic such as a specified pH, salt concentration, surfactantproperty, viscosity and the like. The ability of a phosphomolecule tobind selectively to a phosphoaffinity material can be improved, enhancedand/or stabilized in the presence of sample ingredients such asinorganic salts, alcohols, detergents and surfactants, if desired. In anembodiment of a method of the invention, a sample contacted with aphosphoaffinity material in the presence of a detergent. In a specificembodiment, the detergent is an ionic detergent such as SDS. A varietyof detergents can be used when contacting a sample with aphosphoaffinity material. The detergent can be anion, cationic,zwitterionic or non-ionic. Those skilled in the art will be able toselect a suitable detergent for use with a particular sample andphosphoaffinity material. Example 7 describes use of an ionic detergentduring sample incubation with phosphoaffinity material, and use ofnon-ionic detergent during phosphopeptide elution from thephosphoaffinity material.

The ability of a phosphomolecule to bind selectively to aphosphoaffinity material containing a hydrated metal oxide can bemodulated by pH if desired. In aqueous-based media the predominantsurface functional group on metal oxides is the hydroxyl group. Withoutwishing to be bound by theory, it appears that hydroxyl groups ofhydrated metal oxides are polarized and electrically charged and thisstate allows interaction with phosphorylated molecules under certain pHconditions. The oxide surface adsorbs and/or desorbs protons fromsolution, thus influencing the surface charge. This induceselectrostatic effects in the vicinity of the charged surface, which canaffect the capacity of the metal oxide for sorption of different ionicspecies from the aqueous-based media. At low pH values the surfacecharge becomes positive, while at high pH values it becomes negative.The pH value at which the particles possess no surface charge isreferred to as the isoelectric point or pH of zero zeta potential. Theloss or gain of protons is commonly considered as an acid-base reactionat the metal oxide surface. A variety of different surface hydroxylgroups can be present on a metal oxide surface. When a surface hydroxylgroup is coordinated to a single metal atom, it is referred to as asingly coordinated or terminal hydroxyl group, whereas if the hydroxylgroup is coordinated to two, three or four metal atoms, it is referredto as a bridging hydroxyl group. For iron oxides, the surface hydroxylgroups can be coordinated to one, two or even three underlying metalatoms. Two surface hydroxyl groups can also be bound to a single metalatom. The configurations of the different types of surface groups dependupon the structure of the oxide and the crystal face being examined,with different surface groups likely to display different chemicalproperties. Adsorption of phosphorylated molecules occurs by complexformation reactions between the dissolved phosphorylated solute and thetitratable surface functional groups of the hydrated metal oxidesurface. Those skilled in the art will be able to empirically confirm anappropriate pH for use of a particular hydrated metal oxide.

In the case of titanium dioxide surfaces, protonation and consequentlypositive charge attributes are achieved below the material's isoelectricpoint of approximately 6.0. Other metal oxide surfaces differ in theisoelectric point that is conducive to the generation of a propersurface for affinity capture of phosphorylated molecules. For example,hydrated zirconia possesses an isoelectric point of 8.2, while hematite,yttrium oxide, and gibbsite possess isoelectric point values of 7.5,8.5, and 10.0, respectively. Inclusion of certain alkali metals in themedia can shift the isoelectric point of hydrated metal oxide particlesto higher pH values, as observed with rutile particles incubated withbarium, calcium or magnesium salts. The isoelectric point of hydratedmetal oxides can also be shifted depending on the preparation method,trace impurities and the degree of hydration, and the like. Overall,metal oxides of the general formula Me₂O₃ typically have isoelectricpoints of about 9.0. Metal oxides of the general formula MeO₂ typicallyhave isoelectric points that increase with the metal atom ionic radiusand with decreasing electronegativity of the metal atom. These factorscan be useful to those skilled in the art for determining conditions forisolating and/or detecting phosphomolecules.

For example, the isoelectric point of the hydrated metal oxide canestablish an upper limit for pH of a sample solution used when forming aphosphomolecule-phosphoaffinity material complex. Reciprocally, elutioncan be favored at pH values above the isoelectric point of the hydratedmetal oxide. When using membranes or filters containing amorphous orweakly crystallized hydrated metal oxides, strongly acidic (pH<3.0) andstrongly basic (pH>11.0) solutions can cause chemical instability of thehydrated metal oxide surface. Those skilled in the art will be able toconfirm the integrity of membranes, filters and other supports processedunder various pH conditions.

The adsorption of alkali or alkaline-earth metal cations can be usefulfor regulating binding of a hydrated metal oxide to a phosphomolecule.Exemplary metal cations useful for this purpose include Ba (II), Mg(II), and Ca (II).

In certain embodiments of the present invention, a phosphomoleculeisolated and/or detected using a method of the invention is contained ina sample solution. In other embodiments of the present invention, thephosphomolecule is contained on a support. If desired, one or more typesof phosphomolecule can be presented on a support while other types arepresented in solution. Methods for attaching a phosphomolecule to asupport are well known to those skilled in the art, and are exemplifiedherein below.

Methods described herein can involve separating aphosphomolecule-phosphoaffinity material complex from a sample, therebyisolating the phosphomolecule from the sample. Separation of thephosphomolecule-phosphoaffinity material complex can be achieved by avariety of well known means. In certain embodiments, a phosphomoleculeand/or a sample (i.e. a phosphomolecule contained in the sample) can beattached to a support. When a portion of aphosphomolecule-phosphoaffinity material complex is attached to asupport, a separation can be performed by removing a liquid phase fromthe support and/or by washing the support to remove a liquid phase, gel,colloidal, or other type of non-liquid phase from the support. Theseparation can occur in a variety of formats, such as column, membrane,particle separation by gravity, vacuum, magnetic or other force, and thelike. Similarly, in a certain embodiment, the hydrated metal oxide is aparticle, and separation can be carried out by a particle separation.Alternatively, separation can be performed by collecting thePhosphomolecule-phosphoaffinity material complex, or a portion thereof,using a binding partner such as an antibody, ligand, receptor, antigen,complementary sequence, and the like, which selectively binds to anepitope within the phosphomolecule-phosphoaffinity material complex.

Methods described herein can involve detecting formation of aphosphomolecule-phosphoaffinity material complex in order to detect thepresence of the phosphomolecule in a sample. Procedures for detectinginteraction between molecular entities are well known to those skilledin the art. Such detection procedures generally involve detecting aphysicochemical change in at least one of the interacting entities, ordetecting the presence of one of the interaction entities when thepresence of the other entity is known. A phosphomolecule-phosphoaffinitymaterial complex or portion thereof can be detected by observing aphysiochemical property as well as by observing a functional activity. Aphysicochemical property such as mass, fluorescence absorption,emission, energy transfer, polarization, anisotropy, and the like, canbe observed without chemical modification of thephosphomolecule-phosphoaffinity material complex, if desired.Alternatively, the complex or a portion thereof can be subjected to sometype of chemical modification that facilitates detection of aphysicochemical property. A functional property such as interactioncapability, enzymatic activity and the like can be observed bycontacting the phosphomolecule-phosphoaffinity material complex orportion thereof, with an appropriate binding partner, for example anantibody, antigen, receptor, ligand, co-factor, subunit, complementarysequence, substrate and the like.

Exemplary well known methods for detecting molecular complexes andcomponents thereof include measurements of absorbance, transmission,mass, charge to mass ratio, fluorescence intensity, fluorescencepolarization, time-resolved fluorescence, resonance light scattering,surface-enhanced Raman scattering, electron paramagnetic resonance,refractive index absorbance, nuclear magnetic resonance,microcalorimetry, surface plasmon resonance, refractive index changes,spectropolarimetry, ellipsometry and a variety of spectroscopiccharacteristics such as those measurable by inductively-coupled plasmamass spectrometry, Fourier transform infrared spectrometry, and atomicabsorption spectrometry.

When a phosphomolecule-phosphoaffinity material complex, or componentthereof, contains a luminescent or dye component, detection can be byvisual observation on a UV transilluminator, or by using a UV-basedcharged coupled device (CCD) camera detection system, a laser-based gelscanner, a xenon-arc-based CCD camera detection system, a Polaroidcamera combined with a UV-transilluminator as well as a variety of otherdevices used for detecting luminescence.

A phosphoaffinity material useful as a detection agent can have avariety of physical forms. For example, when in particle form, thephosphoaffinity material can be bound to the phosphomolecule anddetection can be achieved by detecting a physiocochemical property ofthe particle or the interaction of the particle with a phosphomolecule.Examples 10 and 13 describe detecting a hydrated metal oxide portion ofa phosphomolecule-phosphoaffinity material complex using fluorescenttagging of the hydrated metal oxide. Example 11 describes detectinginteraction between a phosphomolecule and a phosphoaffinity materialduring formation of a phosphomolecule-phosphoaffinity material complexby fluorescence resonance energy transfer. Example 12 describesdetecting a metal portion of a phosphomolecule-phosphoaffinity materialcomplex using inductively coupled mass spectrometry.

As a further non-limiting example of a method for detecting aphosphomolecule-phosphoaffinity material complex, binding of hydratedmetal oxide particles to phosphorylated substrates on solid supports canbe detected by methods similar to those employed in the detection ofsilver and gold particles by Resonance light scattering (RLS)techniques. RLS particles have been used as labels for analyte detection(Ygueyabide and Ygueyabide, 2001). RLS particles are ultra-sensitivelabels that have been implemented in a wide range of analyticalbioassays. Spherical gold and silver RLS particles of uniform dimensionranging between approximately 40 and 120 nm diameter generate intensemonochromatic scattered light when illuminated with a narrow beam ofwhite light. The scattered light signal generated by a single RLSparticle is roughly 10⁴ to 10⁶ times greater than the signal obtainedfor a conventional small molecule fluorophore and relatively easilydetected by dark field illumination. The intensity and color of thescattered light generated by individual RLS particles is photostable anddependent upon the particle's composition and diameter. The surface ofRLS particles can be derivatized with a variety of functionalities toinduce selective binding in analytical assays. Sensitive RLS reagent andinstrumentation systems for microarrays, immunocytology/histology, insitu hybridization, microtiter well assays and microfluidics have beendeveloped. Particles made of materials other than cold and silver can beused for an RLS technique if desired. For example,polystyrene-polyacrylic acid particles have been employed in RSLexperiments with proteins (Wang et al, 2004). A hydrated metal oxideparticle or phosphoaffinity particle including a surface of hydratedmetal oxide can be used with RLS assays to detect a phosphomolecule orphosphomolecule-phosphoaffinity material complex.

Other properties of hydrated metal oxide particles can be useful fordetecting phosphomolecules and phosphomolecule-phosphoaffinity materialcomplexes. Biosensors, also known as label-less detection systemsbecause of their direct detection of binding without fluorescent orother radioactive labels, detect mass of entities bound to recognitionmolecules immobilized on a solid support. Some biosensor methods, suchas resonant cantilevers, surface acoustic wave sensors and the like,detect mass binding directly. The density of titanium dioxide is about 4grams per cubic centimeter, significantly higher than that of protein,making it a suitable mass label for this type of sensing. Other sensors,known as optical biosensors, detect changes in refractive index in alocal binding area of the sensor. The local refractive index increasesas the density of higher index molecules and particles bind to thesurface. For example, titanium dioxide exists in two common crystallineforms, anatase with a refractive index of 2.49 and rutile with arefractive index of 2.903, compared to a refractive index of about 1.45for protein and 1.33 for aqueous buffer. Titanium dioxide therefore canbe a sensitive label for optical biosensors. Common optical biosensorsinclude surface plasmon resonance (SPR), evanescent waveguide andcolorimetric resonant reflective devices. Finally, surface-enhancedRaman scattering can be useful for ultrasensitive detection, includingsingle-molecule detection. Crystals of Ag—TiO₂, for example, can beuseful for detecting phosphomolecules andphosphomolecule-phosphoaffinity material complexes by surface-enhancedRaman scattering.

For use as a detection agent, a phosphoaffinity material can be labeledor associated with a detectable tag. For example, a hydrated metal oxidecan be labeled with a dye before or after formation of thephosphomolecule-phosphoaffinity material complex. As a specific example,when a metal oxide particle, such as a nanoparticle, is used as thephosphoaffinity material, the nanoparticles can be allowed to bind to aphosphomolecule and can then be labeled with a dye. This can beaccomplished, for example, by generating a mordant dye lake (Kornblumand Lopez, 1970). A lake is the water insoluble form of a dye (Marshalland Horobin, 1973; Wou and Mulley, 1988; Lillie et al, 1976; Meloan etal, 1973; Ishikawa et al, 2003). Strong attractive forces betweenhydrated metal oxides or freshly precipitated salts (such as calciumsulfate or barium sulfate) and certain dye molecules are thought toresult in their co-precipitation. Lakes are generally more stable thandyes and are considered ideal for coloring products containing fats andoils or items lacking sufficient moisture to dissolve dyes. A range ofdyes representing diverse structural classes can be suitable forhighlighting inorganic metal oxide particles in this way, includinganthracene, azo, indigoid, diaryl methane, triaryl methane, oxyketone,acridine, azine, oxazine, thiazine, quinoline, polymethine, hydrazone,triazene, porphyrin, porphyazin, quinacridones, formazane nitro, sulfur,nitroso quinone imide, azaphilone, cyanine and azomethine. Non-limitingexamples of specific dyes include rhodamine B, rhodamine 6G, acidalizarin violet, morin, tetrahydroxyflavanol,2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole, 2-hydroxyterephthalate,2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid,2-[6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid,N-tert-butyl-phenylnitrone, 2-methyl-2-nitrosopropane-dimer, TEMPO-9-AC,proxyl fluorescamine and coumarin-3-carboxylic acid. Dye familiesincluded those listed above are described, for example, in Handbook ofFluorescent Probes and Research Products, Ninth Edition by Dr. RichardP. Haugland (Molecular Probes, 2003).

Certain particles of metal oxides, such as titanium dioxide, are capableof photocatalytic oxidation of dyes (Marci et al, 2003; Saquib andMuneeer, 2003). Photocatalysts, such as Degussa P25 and UV100 are knownto facilitate this reaction. A photocatalytic oxidation reaction can beused for detecting a phosphorylated molecule. Since primarily hydroxyderivatives of organic compounds are identified in aqueous suspensionsof titanium dioxide that have been irradiated with UV light, themechanism of degradation is thought to be based upon hydroxyl radicalattack. Fluorophores, such as 2-hydroxyterephthalate,2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and2-[6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) can beemployed to selectively detect the highly reactive oxygen species (hROS)generated, such as hydroxyl radicals. In addition, bothN-tert-butyl-phenylnitrone and 2-methyl-2-nitrosopropane dimer formrelatively stable free radicals that can be detected by their electronparamagnetic resonance. TEMPO-9-AC and proxyl fluorescamine are twoadditional fluorogenic probes for detecting hydroxyl radicals. Both ofthese molecules contain a nitroxide moiety that quenches theirfluorescence. However, once TEMPO-9-AC or proxyl fluorescamineencounters a hydroxyl radical or superoxide, its fluorescence isrestored and the radical's spin signal is reduced, making these probesuseful for detecting radicals either by fluorescence or by electron spinresonance spectroscopy. The succinimidyl ester of coumarin-3-carboxylicacid (SECCA) is another useful fluorogenic reagent for detectinghydroxyl radicals. This amine-reactive reagent which can be coupled to awide variety of molecules, is converted by hydroxyl radicals to a highlyfluorescent 7-hydroxycoumarin adduct. Detection of phosphomolecules byhydroxyl radical formation can be performed on a range of solid orsemisolid surfaces, including polymeric membranes, polyacrylamide gels,agarose gels, microarrays or polymeric beads.

The methods of the invention for isolating and/or detecting aphosphomolecule can be carried out in a variety of multiwell plate-basedformats, if desired. Such methods are exemplified in FIGS. 1 to 4. FIG.1 depicts a relatively simple fractionation approach, utilizing hydratedmetal oxide particles that are suspended above a porous support,positioned at the bottom of the wells of a multiwell microplate device.In the schematic, a filtration device is shown generically. Three wellsof the multiwell plate are depicted in the diagram. One plate suitablefor this approach is the Millipore MultiScreen 96-well filtration plate,containing Durapore 0.65 μm membrane. The device includes a separationmultiwell structure or plate (1) containing separation plate wells (2),output apertures (3) and porous supports (4). Hydrated metal oxideparticles (5) are entrapped on top of the porous supports. The particlesserve as the affinity matrix for capturing the phosphorylated molecules.Also depicted in the diagram is a wash receiving multiwell structure orplate (7) including wash receiving plate wells (8). The incubation andbinding is illustrated in (A). During the incubation and binding (A),samples suspected of containing phosphorylated molecules are prepared inbinding buffer (6) and then incubated in the wells of the separationmultiwell structure or plate (1). Subsequently, the multiwell device ispositioned on a vacuum manifold and samples are filtered (8-18″ Hgpressure) to remove any unbound material. The washing is illustrated in(B). A wash buffer (9), typically the binding buffer, is applied to thewells of the microplate device (1) and subsequently filtered through thefiltration device using a vacuum manifold. The wash product (10) iscollected in the receiving multiwell structure or plate (7). The processof washing can be performed iteratively, as indicated by the curvedarrows linking diagram (A) to diagram (B). That is, the wells can berefilled with fresh binding buffer and the particles are washed one ormore times to reduce contaminants. The elution is depicted in (C). Anelution buffer (11) is applied to the wells of the separation multiwellstructure or plate (1), resulting in the release of the phosphorylatedmolecules from the hydrated metal oxide-coated particles. Finally, theeluent with phosphorylated molecules (12) is collected by vacuum intothe eluent receiving multiwell structure or plate (13). The finalproduct is depicted in (D). The eluent receiving multiwell structure orplate (13) now has the eluent containing the phosphorylated molecules(12). This material can be used, either directly or after furtherprocessing and sample cleanup, for a variety of downstream assays,including protein or peptide characterization and identificationprocedures. Alternatively, the phosphorylated molecules can be eluteddirectly into resin designed to desalt samples, such as pipette tips ormultiwell plates filled with reverse-phase packing (Millipore Zip-TipC18 resin) or anion-exchange packing. For certain downstreamapplications, elution can not be preferable and assays can be performeddirectly on the hydrated metal oxide particles themselves.

FIG. 2 depicts a more advanced fractionation device, including compositemembranes with cellulose or polyvinylidene difluoride core and hydratedmetal oxide surface coating. In the schematic, a filtration device isshown generically. Three wells of the multiwell plate are depicted inthe diagram. The device includes a separation multiwell structure orplate (14) containing separation plate wells (15), output apertures (16)and hydrated metal oxide-cellulose composite membrane (17). Thecomposite membrane serves as the affinity matrix for capturing thephosphorylated molecules. A single membrane or stacks of membranes canbe incorporated into the multiwell structure or plate. Also depicted inthe diagram is a wash receiving multiwell structure or plate (19) thatincludes wash receiving plate wells (20). The incubation and binding isillustrated in (A). During the incubation and binding (A), samplessuspected of containing phosphorylated molecules are prepared in bindingbuffer (18) and then incubated in the wells of the separation multiwellstructure or plate (14). Subsequently, the multiwell device ispositioned on a vacuum manifold and samples are filtered (8-18″ Hgpressure) to remove any unbound material. The washing is illustrated in(B). A wash buffer (21), typically the binding buffer, is applied to thewells of the separation multiwell structure or plate (14) andsubsequently filtered through the filtration device using a vacuummanifold. The wash product (22) is collected in the receiving multiwellstructure or plate (19). The process of washing can be performediteratively, as indicated by the curved arrows linking diagram (A) todiagram (B). That is, the wells can be refilled with fresh bindingbuffer and the composite membranes washed one or more times to reducecontaminants. The elution is depicted in (C). An elution buffer (23) isapplied to the wells of the separation multiwell structure or plate(14), resulting in the release of the phosphorylated molecules from thehydrated metal oxide-coated particles. Finally, the eluent withphosphorylated molecules (24) is collected by vacuum into the eluentreceiving multiwell structure or plate (25). The final product isdepicted in (D). The eluent receiving multiwell structure or plate (25)now has the eluent containing the phosphorylated molecules (24). Thismaterial can be used, either directly or after further processing andsample cleanup, for a variety of downstream assays, including protein orpeptide characterization and identification procedures. Alternatively,the phosphorylated molecules can be eluted directly into resin designedto desalt samples, such as pipette tips or multiwell plates filled withreverse-phase packing (Millipore Zip-Tip C18 resin) or anion-exchangepacking. For certain downstream applications, elution can not bepreferable and assays can be performed directly on the compositemembranes themselves.

FIG. 3 depicts an alternative approach to retrieving phosphorylatedmolecules using polystyrene multiwell microplates in which the wells ofthe device have been coated with hydrated metal oxides. In theschematic, a multiwell microplate device is shown generically. Threewells of the multiwell plate are depicted in the diagram. The deviceincludes a separation multiwell structure or plate with hydrated metaloxide coating on the well walls (26) containing separation plate wells(27). The hydrated metal oxide coating (28) serves as the affinitymatrix for capturing the phosphorylated molecules. The incubation isillustrated in (A). During the incubation (A), samples suspected ofcontaining phosphorylated molecules are prepared in binding buffer(29)and then incubated in the wells of the separation multiwell structure orplate (26). The binding is depicted in (B). Captured phosphorylatedmolecules (30) are bound to the hydrated metal coating (28), leaving thesample binding buffer depleted of phosphorylated molecules (31). Thewashing is depicted in (C). The sample buffer depleted of phosphorylatedmolecules is removed using a manual or automated pipetting device (32)and wash solution (33), typically the binding buffer, is added back tothe wells. This washing can be repeated one or more times. The finalproduct, bound to the walls of the separation multiwell structure orplate with hydrated metal oxide coating is depicted in (D). Thephosphorylated molecules (30) are retained on the walls of the wells forsubsequent assay, though an elution buffer can be applied, resulting inthe release of the phosphorylated molecules from the walls, and then thephosphorylated molecules can be recovered by manual or automatedpipetting.

FIG. 4 depicts another approach to retrieving phosphorylated moleculesusing multiwell microplates and inorganic composite materials. In thisinstance, particles with a magnetic core and hydrated metal oxide shellare employed. In the schematic, a multiwell microplate device is showngenerically. Three wells of the multiwell plate are depicted in thediagram. The device includes a separation multiwell structure or plate(34) containing separation plate wells (35), and magnetic particles withhydrated metal oxide-coating (36). The composite particle serves as theaffinity matrix for capturing the phosphorylated molecules. Theincubation and binding is illustrated in (A). During the incubation andbinding (A), samples suspected of containing phosphorylated moleculesare prepared in binding buffer (37) and then incubated in the wells ofthe separation multiwell structure or plate (34). The binding of thephosphorylated molecules to the magnetic particles and subsequentseparation of the particles from the bulk solution with a magneticseparator is shown in (B). The magnetic particles with capturedphosphorylated molecules (39) are concentrated at the bottom of thewells (40) with the aid of a magnetic separator (41). In the process thebinding buffer (38) is depleted of phosphorylated molecules. The washingis depicted in (C). The sample buffer depleted of phosphorylatedmolecules is removed using a manual or automated pipetting device (42)and wash solution (43), typically the binding buffer, is added back tothe wells. This washing can be repeated one or more times. The elutionis depicted in (D). An elution buffer (45) is applied to the wells ofthe separation multiwell structure or plate using a manual or automatedpipette (44), resulting in the release of the phosphorylated moleculesfrom the magnetic particles. The collection of the final product isdepicted in (D). The eluted phosphorylated molecules in solution (45)have been drawn up using a manual or automated pipette (44) and cansubsequently be dispensed for a variety of downstream assays, includingthose devised to characterize or identify the phosphorylated molecules.The phosphorylated molecules can be dispensed directly into resindesigned to desalt samples, such as pipette tips or multiwell platesfilled with reverse-phase packing (Millipore Zip-Tip C18 resin) oranion-exchange packing. If desired, elution need not be performed whenassays can be performed directly in a multiwell microplate.

As is described above, a method of the invention can involve filtering asample in the presence of a phosphoaffinity material, such as particlesor membranes. The viscosity of aqueous-based suspensions of particlescan change by several orders of magnitude as a function of solution pH.For example, solutions containing 30% anatase have the viscosity ofwater far from the particle isoelectric point, and the viscosity ofmolasses near the isoelectric point. When applying samples containingdetergents or surfactants to filtration devices, sample foaming canoccur during filtration. Such foaming can cause sample handling problemsand contribute to cross-contamination among sample wells. Foaming can bereduced by placing samples in solutions containing anti-foaming agents,such as organic solvents, for example methanol or acetonitrile. Anexemplary sample solution includes 60% methanol or acetonitrile, 40%water containing 0.1% formic acid or 60% methanol or acetonitrile, 40%50 mM ammonium carbonate, pH 8.0. Choice of sample solution can takeinto consideration the isoelectric point of the particular hydratedmetal oxide of a selected phosphoaffinity material. The final proteinconcentration in the sample solution can typically be 0.05-5 mg/ml, suchas about 0.4-0.6 mg/ml, although higher or lower protein concentrationscan be used. Extraction and solubilization of samples can be facilitatedby intermittent vortexing and sonication, if desired. When selecting asolubilization material for use in a method described herein, theinteraction of the solubilization material with a selected analyticalmethod can be taken into consideration. For example, surfactants canreduce peptide ionization in mass spectrometry and interfere withchromatographic separations such as reversed-phase liquidchromatography, while solutions containing organic solvents can be morecompatible mass spectrometry and liquid chromatography. A bufferedorganic solvent can be useful for solubilizing and isolating a varietyof proteins, including integral membrane proteins, such as proteinscontaining transmembrane-spanning helices.

The invention provides commercial packages useful for carrying out amethod for isolating and/or detecting a phosphomolecule as describedherein. A commercial package of the invention contains a phosphoaffinitymaterial incorporating a metal oxide, or reagents useful for formingsuch a material. A commercial package of the invention can contain avariety of components in addition to a phosphoaffinity material. Apackage can contain, for example, instructions for preparing aphosphoaffinity material; for using a phosphoaffinity material forisolating a phosphomolecule; for using a phosphoaffinity material todetect a phosphomolecule, or a combination of instructions. Instructionsoptionally can include a recommendation regarding the concentration ofsample for use in a particular application, as well as guidanceregarding temperature, buffer conditions and incubation time periods. Acommercial package of the invention optionally can contain othercomponents, such as one or more protein or peptide fractionationdevices, labeled polypeptides, fluorescent dyes, binding buffers, washbuffers, molecular weight standards, isoelectric point standards,phosphorylation standards, fixatives, stains, antibodies, lectins,aptamers, phosphatase substrates, kinase substrates, detection reagents,magnetic separator, and the like. Those skilled in the art will be ableto select suitable components for inclusion in a commercial package ofthe invention based on such exemplary factors as design of the assayprotocol, the particular phosphoaffinity material used for detection orisolation, method of measurement to be employed once the assay has beenperformed, consumer price point, shipping and handling suitability andthe like.

In particular embodiments, the phosphoaffinity material includes asupport. Exemplary supports include membranes, particles, matrices,spin-columns, microcolumn pipette tips, multi-well microwell strips, andmulti-well microplates. Specific examples of phosphoaffinity materialsinclude filtration devices including membranes and filters containingone or more porous or semi-porous hydrated metal oxide surfaces and/orcoatings; filtration devices containing filters, particles and/ormembranes that contain or incorporate hydrated metal oxides as a coatingon fiber surfaces, entrapped within the membrane's polymeric matrix orpores or presented as a layer on top of the membrane; and filtrationdevices configured as spin columns, microcolumn pipette tips, multi-wellstrips, and/or multi-well microplates.

The invention provides a variety of commercial packages useful forcarrying out a method for isolating and/or detecting a phosphomoleculein a sample. In an embodiment, a commercial package includes a hydratedmetal oxide attached to a support, wherein the hydrated metal oxidecomprises yttrium. In another embodiment, a commercial package includesa phosphoaffinity unit, the unit comprising a plurality of supportsheets coated with a hydrated metal oxide. As used herein, the term“phosphoaffinity unit” means a device that contains a phosphoaffinitymaterial, such as a sample receptacle, column, plate and the like. Thesupport sheet can be, for example, a membrane or paper, such ascellulose. In a further embodiment, a commercial package contains aphosphoaffinity particle comprising a hydrated metal oxide and adetectable agent that binds to the hydrated metal oxide. In anembodiment, a commercial package of the invention includes aphosphoaffinity material comprising titanium dioxide particles, and adetectable agent that binds to titanium dioxide particles. In a specificembodiment, the titanium dioxide particles are crystals of titaniumdioxide. In another embodiment, the titanium dioxide particles are aparticle support coated with titanium dioxide.

The invention provides a composition comprising a complex of aphosphoaffinity material and a phosphomolecule. The complex can be used,for example, as a standard, control, binding partner for a known orunknown substrate or analyte, and the like.

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are also includedwithin the definition of the invention provided herein. Accordingly, thefollowing examples are intended to illustrate but not limit the presentinvention.

EXAMPLE 1

This example describes isolation of phosphoproteins by selective bindingwith yttrium oxide, yttrium iron garnet or titanium dioxide.

Affinity columns for isolating phosphopeptides were prepared as follows:15-25 mg of yttrium oxide (Aldrich catalog #205168-10), yttrium irongarnet (Aldrich catalog #634417-10) or titanium dioxide (Aldrich catalog#634662-25) particles was packed into five inch disposable columns(Evergreen Scientific, Los Angeles, Calif.). Each column was washed with2×1 ml of 1% (v/v) formic acid in deionized water. The contents of thecolumns were mixed intermittently using a vortex mixer. Each column wasthen washed with 3×1 ml of binding buffer (0.5 M sodium acetate, 0.2 Msodium chloride pH 5.5). The contents of the columns were mixedintermittently using a vortex mixer.

Phosphoproteins were isolated as follows: one ml of a solution (0.2μg/μl, total protein) containing both bovine carbonic anhydrase II andchicken ovalbumin (˜1:1 ratio) in binding buffer was added to eachcolumn. The suspension was intermittently mixed for one minute using avortex mixer. The columns were capped on both ends and the particlesuspension was allowed to incubate in a horizontal position for one hourwith agitation, using a Nutator mixer. The columns were placed in avertical position, drained and packed, and the hydrated metal oxideparticle beds were rinsed with 3 volumes of 0.5 ml binding buffer. Thecolumns were then washed with 3 volumes of 0.3 ml elution buffer(phosphate-buffered saline, pH 7.4). The first elution wash wasequilibrated in the column for one hour before draining. The combinedeluents were collected in a 15 ml centrifuge tube. Proteins present inthe elution buffer were precipitated by addition of ice cold acetone toa total volume of 12 ml, vortex mixing and centrifuging for fiveminutes. The acetone was removed and the resulting white solid wasdissolved in 20 μl of 10 mM Tris HCl, 2% (v/v) SDS, pH 7.6 prepared indeionized water and 30 μl of sample dilution buffer. The sample dilutionbuffer was prepared by combining 0.1 ml of 0.5 M dithiothreitol inethanol, 0.9 ml of 10 mM Tris HCl, 2% (v/v) SDS, pH 7.6 in deionizedwater and 1 ml of NuPage LDS sample buffer 4× (Invitrogen Corporation,Carlsbad, Calif.). One ml of bovine carbonic anhydrase/chicken ovalbumincontrol protein sample (˜1:1) (0.2 μg/μl total protein) in bindingbuffer was also acetone precipitated directly to serve as a referencestandard for monitoring the enrichment of the phosphorylated protein(ovalbumin) relative to the unphosphorylated protein (carbonicanhydrase).

Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis,according to standard procedures. Bio-Rad Broad Range molecular weightmarkers were included in some lanes on the gels to facilitateidentification of serum albumin and ovalbumin based on molecular weight.After electrophoresis, the gels were fixed, stained with SYPRO Rubyprotein gel stain (Molecular Probes/Invitrogen, Eugene, Oreg.),destained and fluorescent signal imaged using the ProXPRESS 2D proteomicimaging system (PerkinElmer LAS, Boston, Mass.).

Representative line traces obtained from resultant protein samplesprocessed on yttrium oxide, yttrium iron garnet or titanium dioxide areshown in FIGS. 5 to 7, respectively. The profiles of the referencestarting materials are shown in part A of the figures, while theisolated material is depicted in part B of the figures. The X-axis ofthe figures represents the distance traveled by the proteins in theelectrophoresis gel, while the Y-axis corresponds to the fluorescenceintensity of the total protein stain.

In FIGS. 5 to 7, peaks identified as (46), (49) and (52) correspond tofluorescently stained carbonic anhydrase bands, while (47), (48), (50),(51), (53) and (54) correspond to fluorescently stained ovalbumin bands.These data show that ovalbumin (the phosphorylated protein) wasseparated from carbonic anhydrase (the non-phosphorylated protein) usingaffinity columns prepared with particles of yttrium oxide, yttrium irongarnet and titanium dioxide.

EXAMPLE 2

This example describes isolation of phosphopeptides using a titaniumdioxide phosphoaffinity material in a 96-well filtration plate format.

The phosphoaffinity material in 96-well filtration plate format wasprepared as follows: about 0.2 g of titanium dioxide particles wasactivated in 10 ml of 1% formic acid for 10 min at room temperature andwashed with deionized water several times using centrifugation tocollect the beads. The pre-washed beads were re-suspended in 10 ml ofdeionized water and 200 μl/well of the resulting suspension was pipettedinto the 96-well MULTISCREEN DV (Millipore Corporation, Bedford, Mass.)plate. The resulting titanium dioxide loaded plate was pre-equilibratedby washing wells in 500 mM sodium acetate pH5.5+200 mM NaCl buffer(Binding Buffer).

Isolation of phosphopeptides was performed as follows: about 200 μl/wellof the 2.5 μM solution of phosphorylated synthetic peptide and 1 μM ofnon-phosphorylated synthetic peptide was allowed to bind in wells of thetitanium dioxide loaded plate by incubating the particles with peptideson a plate shaker for 15 min at room temperature followed byvacuum-assisted passage of the peptide solution through the titaniumdioxide particles. Different starting concentrations for phosphorylatedand non-phosphorylated peptide were used to balance the peak intensitiesfor these peptides in MS spectra, as phosphorylated peptides are knownto be more difficult to ionize. The material that was unbound to thetitanium dioxide particles was collected as a flow-through fractionconcentrated on a ZipPlate as recommended by the manufacturer. Remainingunbound or non-selectively bound material was washed off the titaniumdioxide particles with ten 180 μl volumes of the Binding Buffer.Peptides bound to the titanium dioxide particles were eluted with 180 μlof 1% ammonium hydroxide solution and concentrated on a ZipPlate asrecommended by the manufacturer. The concentrated peptides were elutedfrom the ZipPlate directly on to a MALDIChip with the 5 mg/mlα-cyano-4-hydroxycinnamic acid+50% acetonitrile+0.1% trifluoroaceticacid solution and analyzed on the prOTOF 2000 mass spectrometer.

Materials used in the above experiments included synthetic p60 c-srcpeptide (521-533)Thr-Ser-Thr-Glu-Pro-Gln-Tyr-Gln-Pro-Gly-Glu-Asn-Leu-COOH (cat #8249),and pp60 c-src peptide (521-533)Thr-Ser-Thr-Glu-Pro-Gln-Tyr(P)Gln-Pro-Gly-Glu-Asn-Leu-COOH (cat #8250)were obtained from SynPep Corporation, Dublin, Calif. The “P” isparentheses indicated phosphorylation of the tyrosine residue. Titaniumdioxide particles of mesh-325 (cat# 248576-100 g), acetonitrile (cat#270717-6X1L), trifluoroacetic acid (cat# 302031-100ML), ammoniumhydroxide (cat# 338818-100 ML), formic acid (cat# PR15225KR), sodiumacetate (cat# 241245-500G), and sodium chloride (cat# 22351-4) werepurchased from Sigma-Aldrich (St. Louis, Mo.). ZipPlates (cat#ZPC180010), 96-well MultiScreen-DV plates (cat# MADVN6510), and customvacuum manifold were obtained from Millipore Corporation (Billerica,Mass.). Polypropylene 96-well collection plates (cat# EK-21261) werepurchased from E & K Scientific Products, Inc. (Los Gatos, Calif.).Alpha-cyano-4-hydroxycinnamic acid (cat# M001) and Peptide calibrationMix 1 (cat# C101) were purchased from LaserBio Labs (Sophia-AntipolisCedex, France). Mass spectrometry (MS) spectra were acquired usingMALDIChip target plates (catalog #N7014021) and a prOTOF 2000 O-TOF massspectrometer (PerkinElmer/SCIEX, Concord, ON, Canada).

FIG. 8, top and bottom, respectively depict MALDI-TOF mass spectrometryprofiles of unphosphorylated c-src peptide (p60 c-src peptide) andphosphorylated c-src peptide (pp60 c-src peptide) spotted directly ontoa MALDIChip plate without fractionation. The monoisotopic peak massescorresponding to the expected peptide masses were shown for each MSspectrum. The ratio of intensities between pp60 c-src peptide and p60c-src peptide peaks was 1.1. FIG. 9 depicts the MALDI-TOF massspectrometry profile of the peptide fraction that was unbound to thetitanium dioxide particles.

These data show that the pp60 c-src peptide (bottom) was retained on thetitanium dioxide particles while the p60 c-src peptide (top) passedthrough the titanium dioxide particles. The ratio of intensities betweenpp60 c-src peptide and p60 c-src peptide peaks is 0.05. FIG. 10 depictsthe MALDI-TOF mass spectrometry profile of the peptide fraction elutedfrom the titanium dioxide particles. The pp60 c-src peptide wasselectively retained and eluted from the titanium dioxide beads. Ascarcely detectable amount of non-phosphorylated peptide was retained onthe titanium dioxide particles. The ratio of intensities between pp60c-src peptide and p60 c-src peptide peaks was 20.

Four independent fractionations and analyses were performed for eachpeptide. The expected peptide masses for p60 c-src peptide and pp60c-src peptide are 1463.0 and 1543.5 daltons, respectively, and can bereadily distinguished on the corresponding MS spectra (FIGS. 8 to 10).Analysis of unbound and bound peptide fractions obtained from thetitanium dioxide particles demonstrates selective retention and recoveryof phosphorylated peptides under the described experimental conditions.The ratio of intensities of the directly analyzed unfractionatedpeptides was close to 1 (FIG. 8), indicating that peptide ionizationresponses were balanced under the experimental conditions and can becompared to one another. Taken together, these data indicate that about20-fold more pp60 c-src peptide was retained and eluted on titaniumdioxide particles than p60 c-src peptide.

EXAMPLE 3

This example describes isolation of phosphoproteins using a titaniumdioxide phosphoaffinity material, further fractionation using gelelectrophoresis and detection using mass spectrometry.

A sample enriched for phosphoproteins can be prepared using a titaniumdioxide phosphoaffinity material, as is described above in Examples 1and 2. The sample is then separated on an SDS-polyacrylamide gel usingstandard methods. Gels are stained using SYPRO Ruby protein gel stainper manufacturer's suggestions and fluorescent signal is imaged using aProXPRESS 2D proteomic imaging system or similar gel imaging device. Thegel is scanned with a 460/80 nm excitation band pass filter and 650/150nm emission band pass filter. Fluorescent spots are then excised fromthe gel. For destaining, the spots are placed into a 1.5 mLmicrocentrifuge tube and destained with 100 μL 50% methanol, 5% aceticacid for 30 min, 100 μL 0.1% trifluoroacetic acid for 30 min, 100 μL 50%methanol/5% acetic acid for 30 min, and finally dehydrated in 100 μL100% acetonitrile for 10 min. The pieces are air dried before reductionand alkylation. If the proteins have already been reduced and alkylatedbefore the 2-D gel electrophoresis, the following alkylation andreduction steps can be omitted.

For the alkylation and reduction of cysteine residues, enough 20 mMdithiothreitol (DTT) is added to a solution of 0.1 M NH₄HCO₃ tocompletely cover the dried gel pieces (˜50 μL). It can be necessary toadd more solvent as the gel pieces re-swell. Gel pieces are incubated at56° C. for 1 hr. The DTT solution is removed and an equal volume (50 μL)of 100 mM iodoacetamide in 50 mM NH₄HCO₃ is added. The gel pieces areincubated at room temperature in the dark for 30 min., the supernatantis discarded and the gel pieces are washed twice with 100 μL 0.1 MNH₄HCO₃ for 15 min. with occasional vortexing to wash out excessreagents. To extract any excess reagents, the gel pieces are washed with100 μL of 0.1 M NH₄HCO₃/50% ACN for 15 min with occasional shaking. Thesupernatant is discarded and the gel pieces are washed with 100%acetonitrile. The supernatant is discarded and the gel pieces arethoroughly dried in air.

For in-gel digestion with trypsin, a fresh solution of 0.05 mg/mlmodified trypsin in 50 mM NH₄HCO₃/10% acetonitrile is prepared. Thereagent is kept on ice if not used immediately. 10 μL of the freshtrypsin solution is added and the gel pieces are allowed to soak up thetrypsin solution before proceeding to the next step. This typicallyrequires about 10 min. of incubation. The gel pieces are fullyre-swelled by adding 20 μL of 50 mM NH₄HCO₃/10% acetonitrile (finalvolume is roughly 30 μL) and incubated overnight at 37° C.

To extract the peptides, proteolytic digestion is terminated by adding 1μL of 10% trifluoroacetic acid for 10 min. at room temperature. Thesample is mixed using a Vortex, mixer, briefly centrifuged and thesupernatant is removed by aspiration and then placed in a 0.5 ml microtube. 50 μL of 0.1% trifluoroacetic acid is added to the gel pieces andincubation is carried out for an additional 30 min. This solution ismixed and spun, and then the resulting supernatant is combined with thefirst one. 50 μL 60% acetonitrile/0.1% trifluoroacetic acid, is added tothe gel pieces and incubated for 30 min. After mixing, spinning, andcombining with first supernatant in the tube the combined supernatantmixture is dried in a Speed-Vac vacuum concentrator and dissolved againin 10 μL 10% acetonitrile/0.1% trifluoroacetic acid. The peptide mix isthen optionally desalted and concentrated with a CI8 ZipTip column fromMillipore or spotted directly onto a MALDI-TOF target plate, dependingon the sample concentration. The concentrated peptide mixture is mixedwith 0.5 μL matrix (5 mg/ml α-cyano-4-hydroxycinnamix acid in 50%acetonitrile, 0.1% trifluoroacetic acid) and 0.5 μL of sample is spottedon the target plate. The spot is allowed to air dry and is then analyzedusing a prOTOF 2000 MALDI O-TOF Mass Spectrometer (PerkinElmer, Boston,Mass.) or similar instrument. The protein is subsequently identifiedbased on its characteristic peptide mass profile.

In summary, a phosphoprotein enriched sample prepared using a titaniumdioxide phosphoaffinity material can be subjected to furtherfractionation, such as by gel electrophoresis, prior to analysis by massspectrometry.

EXAMPLE 4

This example describes preparation of a titanium oxide phosphoaffinitymaterial in membrane format.

Multiwell plates containing membranes coated/impregnated with titaniumoxide were prepared as follows: MULTISCREEN 96-well (Millipore Corp.,Bedford, Mass.) plates were cleaned by washing 3 times with 250 μl/wellof absolute ethanol followed by 5 times wash with 250 μl/well ofdeionized water (Milli-Q water, Millipore Corp.). Plates were dried at60° C. for 30 min.

A titanium dioxide coating solution (50 mM Ti fluoride solution) wasprepared in deionized water and adjusted to pH ˜1.8 by weighing 0.31 gof Ti (IV) fluoride in a clean polycarbonate screw-cap container; adding20 ml of water, closing the container, and placing it on a magneticmixer for 60 min to completely dissolve the Ti (IV) fluoride. Veryslowly (˜1 ml/min), 26 ml of an aqueous solution of 0.1% NH4OH wasintroduced with constant mixing to avoid localized precipitation oftitanium dioxide, to obtain a solution having pH close to pH 1.8. Ifneeded, the pH of the solution can be adjusted by addition of more 0.1%NH₄OH. The volume of the Ti (IV) fluoride solution was adjusted to 50 mlfinal volume by adding deionized water.

The coating solution (250 μl/well) was applied to the wells of apre-washed and dried MULTISCREEN 96-well plate. The plate was sealedwith a self-adhesive cover to prevent evaporation of the solution. Theplate was incubated at 60° C. in a convection oven for 2 hours. Titaniumdioxide coating solution was removed by gently tapping the plate upsidedown on a clean absorbing surface, such as a paper towel. The wells werethen rinsed with 250 μl of deionized water. The plate was gently tappedupside down on a clean absorbing surface to remove water from the wells.The wash steps were repeated 3 more times. The plates were then dried at60° C. in a convection oven for 40 minutes. Derivatized plates werestored in a desiccated state until ready for use. Materials used for theabove preparation include MULTISCREEN 96-well plates (Cat# MADVN 6510;Millipore Co.); titanium (IV) fluoride (Cat# 333239-100 g; Sigma); Labware (Cat# 353073; Becton Dickinson); and other reagents were purchasedfrom Sigma.

EXAMPLE 5

This example describes isolation of phosphopeptides using a titaniumdioxide phosphoaffinity material in membrane format.

Phosphopeptides were enriched from a mixture containing phosphopeptide(P2P) and non-phosphorylated peptide (P2). The peptides were asdescribed in the previous samples, p60 c-src peptide (P2) and pp60 c-srcpeptide (P2P). For these studies about 16 μl of 100 μM solutions of P2and P2P in water were mixed together and diluted 1:25 in Binding Buffer(20% acetonitrile+20% isopropanol+0.1% formic acid). One half of theresulting peptide mix solution was split further into 2 wells of thecollection plate and set aside as non-fractionated positive controls.The other half of the peptide mix sample was fractionated on titaniumdioxide coated MULTISCREEN 96-well plate (as prepared in Example 4) asfollows. Equal amounts of the sample were loaded into 2 separate wells.The titanium dioxide plate was placed on top of a collection plate in avacuum manifold and low vacuum was applied (less than 25 mm of Hg),allowing the peptide solution to pass through the titanium dioxidecoated membrane and any unbound material to be collected in thecollection plate for analysis. The positive control samples and theunbound material samples were placed into a vacuum oven at 60° C. forabout 30 min to evaporate the acetonitrile and isopropanol beforeconcentrating the samples on a C18-based ZipPlate (Millipore, Bedford,Mass.). After evaporation of organic solvents, the concentrated sampleswere diluted in 0.1% formic acid to the original volume. The samples ontitanium dioxide coated membrane were washed using 180 μl, of theBinding Buffer. The washing step was repeated 5 times. The samples wereeluted with 300 μl of 100 mM ammonium phosphate pH 8.5, and collected ina collection plate using a vacuum manifold as described above. For theseexperiments, phosphorylated and non-phosphorylated forms of p60 c-src(521-533) peptide (abbreviated P2 and P2P hereafter, respectively) wereobtained from SynPep Corporation, Dublin, Calif. The expected m/z valuesfor P2 and P2P are 1463 Da and 1543 Da, respectively.

The eluted samples were acidified by adding about 30 μl of 10% formicacid. Samples were concentrated on a ZipPlate and eluted directly onto aMALDI Chip (PerkinElmer) in duplicate for analysis on the prOTOFMALDI-TOF mass spectrometer (PerkinElmer/SCIEX).

The results are represented by mass spectra of the non-fractionatedpositive control containing P2 and P2P, bound fraction and non-boundfraction are shown in FIG. 11. FIG. 11A shows presence of P2 (1462.6m/z) and P2P (1542.6 m/z) in the starting mixture. FIG. 11B shows thatP2P (1542.6 m/z) bound to the titanium dioxide phosphoaffinity material,while P2 did not. FIG. 11C shows that P2 (1462.7) was present in theunbound fraction. The non-phosphorylated peptide P2 was detected only inthe unbound fraction while the phosphorylated peptide P2P was recoveredin the eluted fraction. Both peptides can be easily distinguished in thenon-fractionated samples based on their respective masses.

In summary, a phosphopeptide containing sample was enriched forphosphopeptide content by processing on a titanium dioxide coatedmembrane in a filtration format.

EXAMPLE 6

This example describes isolation of phosphopeptides from a complexmixture containing serum, using a titanium dioxide phosphoaffinitymaterial in particulate format.

A complex phosphopeptide mixture was prepared by adding about 400 pmolof phosphorylated (P2P) and non-phosphorylated (P2) synthetic peptidesto 20 μl of human serum (Sigma). Unadulterated serum sample was used asa negative control. The two samples are referred to herein as spiked andnon-spiked samples, respectively.

Both spiked and non-spiked serum samples were pre-fractionated by adding2 volumes of acetonitrile resulting in dissociation of protein boundpeptides and precipitation of serum proteins. Peptides in solution phasewere separated from the precipitated proteins by centrifugation.Pre-fractionated samples were diluted in 0.1% formic acid andisopropanol was added to a final buffer composition matching the BindingBuffer described in Example 5. The samples were processed and analyzedin a similar way as described in Example 5 except that titanium dioxideparticles of mesh −325 were deposited on top of Multiscreen membranesand used as chromatographic media instead of titanium dioxide coatedmembranes.

The results are represented in FIG. 12. FIG. 12A shows mass spectra ofunspiked serum and serum containing P2 and P2P prior to fractionation.FIG. 12B shows mass spectra of fractions that bound to thephosphoaffinity material. FIG. 12C shows mass spectra actions that didnot bind to the phosphoaffinity material. There were no spiked peptidesdetected in serum samples that had not been fractionated on titaniumdioxide particles. Principally non-phosphorylated P2 peptide wasdetected in the fraction unbound to titanium dioxide from the spikedserum sample. In summary, enrichment of the phosphorylated P2P peptidewas observed in the eluted fraction from titanium dioxide particles.

EXAMPLE 7

This example describes isolation of a phosphopeptide from an enzymedigest sample using a titanium dioxide phosphoaffinity material inmembrane format.

Phosphopeptides were isolated from a complex peptide mixture generatedfrom trypsin-digested bovine β-casein. About 100 μg of bovine β-caseinin 100 mM ammonium carbonate buffer pH 8.5 was digested with 5 μg oftrypsin (Sigma) for 16 hours. The enzymatic reaction was stopped byadding formic acid to 3% final concentration. Three samples of caseindigest were prepared by diluting 5 μl of the digest sample with theBinding Buffer (see Example 5) by 20-fold. The resulting samples werefractionated on titanium dioxide coated MULTISCREEN membrane plates andanalyzed following the procedures described in Example 5. Samplesconcentrated on ZipPlate without titanium dioxide fractionation andcasein digest directly spotted on MALDI Chip were used as controls.

The results are represented in FIG. 13. FIG. 13A shows distribution ofpeaks in MS spectra of peptides eluted from titanium dioxide, peptidesunbound to titanium dioxide, and peptides present in the tryptic digestof β-casein in the range of m/z values from 700 to 5000. FIG. 13B showsa detailed representation of variants of phosphorylated peptidesenriched from the trypsin-digested bovine β-casein sample. These resultsindicate that both monophosphorylated, biphosphorylated,triphosphorylated and tetraphosphorylated peptides were isolated usingtitanium dioxide coated membranes.

EXAMPLE 8

This example describes isolation of phosphoproteins from a 5-proteinmixture using a titanium dioxide phosphoaffinity material in membraneformat.

A five protein mixture (P_(mix)5) was prepared by combining equalweights of bovine serum albumin (BSA), ovalbumin, carbonic anhydrase,myoglobin, and β-lactoglobulin. Stock solution of P_(mix)5 was diluted1:10 in Binding Buffer (Example 5). One half of the samples were alsodiluted 1:10 in Binding Buffer containing 1% TRITON X100 to reducenon-specific interactions of proteins with titanium dioxide.

Titanium dioxide isolation of proteins was performed as described abovein the previous examples except that three additional stringency washeswith 200 μl of 50 mM Tris-Acetate pH 7.5+1% Triton X100 were added toreduce non-specific interactions of proteins with titanium dioxide.Also, an additional elution step was introduced in which the ammoniumphosphate buffer used in the initial elution was supplemented with 1%SDS in the second elution. Addition of SDS resulted in more quantitativeelution of proteins. Protein samples were precipitated with acetone andanalyzed on a 12% Bis-Tris mini-gel (Invitrogen). Gels were stainedusing SyproRuby total protein gel stain (Molecular Probes). Proteinbands were quantified and the enrichment ratios of ovalbumin relative toother non-phosphorylated proteins were calculated.

FIG. 14 shows results of these experiments. Proteins in the P_(mix)5included phosphorylated ovalbumin (OVA) and 4 non-phosphorylatedproteins (bovine serum albumin (BSA), carbonic anhydrase (CAH),myoglobin (Myo), and β-lactoglobulin), as are indicated on the SDS-PAGEgel shown as FIG. 14A. Two types of eluted samples arerepresented—samples bound to the titanium dioxide membrane in thepresence of 1% TRITON X100 (TE2) or without it (E2). Ovalbuminenrichment ratio relative to non-phosphorylated proteins was calculatedas a ratio of corresponding band intensities on the gel. Normalizedovalbumin enrichment ratios with respect to BSA, CAH and myo are shownin FIG. 14B. These ratios were normalized to the corresponding ratios inthe unfractionated sample (M5). M=Molecular weight markers;M2=BSA+Ovalbumin binary protein mix. These data indicated that at least8-fold enrichment of phosphorylated ovalbumin was observed usingtitanium oxide coated membranes. Increased enrichment to about 50-dold(on average with respect to BSA, CAH and myo) was observed when sampleswere eluted in the presence of detergent.

EXAMPLE 9

This example describes enrichment of phosphomolecules in an arrayformat.

The combination of multi-well microplates and robotic liquid handlinginstrumentation provides a convenient and powerful technology platformfor high-throughput sample processing in drug discovery and leadcompound screening. The microplate/liquid handler platform permits largenumbers of samples or assays to be processed in a parallel fashion.Polystyrene-based multi-well plates are standard for many applicationsbecause the plastic is easy to mold accurately, displays good rigidityfor handling by robotic systems, and provides the optical clarity forplate reading. Polypropylene plates are commonly employed inapplications involving polymerase chain reaction (PCR) because it has alower binding capacity for proteins and nucleic acids than polystyrene.Additionally, it can be molded thinly in order to improve heat transfer.Multi-well microplates are available with various functional coatings,such as streptavidin for the capture of biotinylated molecules, as wellas with various types of filters incorporated into the base in order tofacilitate purification of various molecules of interest.

Microplate-based phosphoprotein purification can be accomplished asfollows. A protein sample is obtained from the specimen to be analyzed.Delipidation can be used for enrichment of phosphoproteins from complexspecimens, such as cell lysates. Such samples will containphosphorylated molecules other than phosphoproteins, such asphospholipids, that can adversely impact recovery of phosphoproteinsaccording to the present methods and systems. An example of a successfulclean-up method for the preparation of proteins prior to multi-wellmicroplate-based phosphoprotein enrichment is chloroform-methanolprecipitation. For example, 600 microliters of methanol is added to 150microliters (150-300 micrograms) of protein sample and the sample ismixed well using a vortex mixer. Then, 150 microliters of chloroform isadded and the sample is once again mixed thoroughly. Next, 450microliters of deionized water is added and the sample is mixedthoroughly. The specimen is then centrifuged at roughly 12,000revolutions per minute (rpm) using a tabletop microcentrifuge. The upperphase obtained from the resulting two-phase separation is discarded,while care is taken so that the white precipitate that forms at theinterface between the two phases is retained. 450 microliters ofmethanol is then added, the sample is mixed well and once againcentrifuged at 12,000 rpm for five minutes, producing a pellet at thebottom of the tube. The entire supernatant is discarded and the pelletis resuspended in a suitable buffer for the affinity purificationprocedure, as detailed below. Suitable cocktails of protease andphosphatase inhibitors can be employed to preserve the integrity of thebiological sample throughout sample preparation. The described samplecleanup procedure serves only as an example and numerous other methodscan be employed (e.g., delipidation can effectively be performed usingan ion-exchange pre-purification of sample), with choice of proceduredictated somewhat by the nature of the biological sample under study.Biological fluids, culture media, suspension cell cultures, adherentcell cultures, bacteria, plant tissue and animal tissue can requiredifferent sample preparation procedures. For example, solid materials,such as animal tissues, organs or whole organisms, can requireadditional preparation prior to delipidation, such as mechanicaldisaggregation using a blender and/or enzymatic disaggregation usingcollagenase and/or elastase.

Once a suitable sample is prepared, the material is resuspended in anappropriate buffer in order to conduct the affinity fractionation,keeping in mind the isoelectric point of the hydrated metal oxidesurface. For titanium oxide-based separation devices, binding bufferswith pH values less than 6.0, such as pH values of around 5.0 to 5.5,are suitable for the intended application. An example of a suitablebuffer for resuspension of the biological material prior tofractionation on a titanium oxide-based matrix is 0.5 M sodium acetate,0.2 M sodium chloride, pH 5.5. More acidic buffers of pH 3-4 can inducecertain proteins to precipitate from solution, and buffers of even lowerpH values can lead to decomposition of certain titanium oxide supports,such as amorphous or microcrystalline layers. Phosphopeptides can becaptured by the phosphoaffinity material using more acidic buffers thanis optimal for phosphoproteins so long as the hydrated metal oxidematrix is stable under the selected binding conditions. Binding buffersare generally formulated to avoid presence of phosphate orpyrophosphate, since these ions can reduce or even eliminate binding ofthe phosphorylated molecules to the matrix. Other additives can beincluded in the binding buffer in order to facilitate solubilization ofthe proteins. Examples of such additives include Triton X-100, sodiumdeoxycholate, urea, thiourea and sodium dodecyl sulfate. Sampletreatment with nucleases, such as DNAse I or RNAse A, can be useful forcleaving nucleic acids that otherwise can render samples too viscous foroptimal filtration. Addition of kinase and or phosphatase inhibitors canalso be employed for preserving the phosphorylation status of thebiological molecules during isolation.

Enrichment of phosphoproteins or phosphopeptides is achieved using afiltration device, such as a spin column or multi-well microplate,containing a porous or semi-porous hydrated metal oxide surface orcoating. First, the filtration device is typically pre-wetted byapplication of a small amount, about 100 μl, of the binding buffer,though pre-wetting with other media, such as water, or 70% ethanol canbe appropriate in certain instances. After about one or more minutes,the buffer can be removed through vacuum filtration, aspiration,centrifugation or other means. Once pre-wetted, the plate can be keptdamp before filtering to avoid a need for rewetting. Next, sampleprepared in appropriate binding buffer is added to the filtration deviceand allowed to incubate with the hydrated metal oxide affinity supportfor a sufficient period of time to permit interaction of thephosphorylated molecules with the hydrated metal oxide surface. Thesample is removed through vacuum filtration, aspiration, centrifugationor other means. Then the device is washed with additional binding bufferusing the same basic approach. Typically, several volumes of bindingbuffer are repeatedly applied to remove nonselectively associated samplecomponents, leaving the phosphorylated molecules associated with thephosphoaffinity membrane.

After undesired materials have been removed by application of the washbuffer, the phosphorylated polypeptides can be eluted with anappropriately formulated elution buffer. In an analogous manner as withthe binding buffer, choice of the composition of elution buffers can beinfluenced by biophysical constraints related to the affinity supportand by properties of the phosphomolecules. Highly basic elution bufferscan reduce stability of the phosphoaffinity material (particularly incases where the surface is amorphous or microcrystalline) and can causedephosphorylation of phosphomolecules through alkaline-inducedelimination of the phosphate group from phosphoserine andphosphothreonine residues. Under certain circumstances, it can bedesirable to dephosphorylate these residues prior to application to thefiltration device, in order to selectively detect or isolate proteinscontaining phosphotyrosine residues. While eluting at pH values higherthan the isoelectric point of the hydrated metal oxide is one option forrecovering phosphorylated molecules from the matrix, thephosphomolecules can also be competitively eluted from the matrix usingphosphate-containing buffers, such as phosphate-buffered saline, pH 7.4.Once eluted, the phosphomolecules can be applied to a desalting device,such as a C-18 reverse-phase packing or an anion-exchange medium, oralternatively can be precipitated with organic solvent, such as ice-coldacetone, or used directly.

EXAMPLE 10

This example describes detection of a phosphorylated polypeptide insodium dodecyl sulfate (SDS)-polyacrylamide gels.

Described below is an exemplary procedure for detecting a phosphorylatedpolypeptide, such as a phosphoprotein or phosphopeptide in an SDS-PAGEgel, using a phosphoaffinity material. In this case, the phosphoaffinitymaterial is a hydrated metal oxide such as yttrium oxide, yttriumaluminum garnet or titanium dioxide in particle or crystal form. Aprotein sample is separated by SDS-polyacrylamide gel electrophoresisaccording to standard procedures. After electrophoresis, the gel isfixed in an acid- and alcohol-containing solution, such as 50% methanol,10% acetic acid and is then washed in deionized water to remove thefixative solution. The gel is then added to a solution containingphosphoaffinity particles/crystals and incubated for 2 hours toovernight at room temperature with gentle shaking (i.e. ˜50 RPM on anorbital shaker). The solution containing the phosphoaffinity particlescan contain simple alcohols, buffer, salts and/or acids in order tofacilitate binding of the phosphoaffinity particles with thephosphoproteins and minimize nonselective binding to the gel matrix orother anionic macromolecules within the sample. The gels are washed in abuffer of similar composition as that used during the binding event,except that phosphoaffinity particles are absent from the solution.Alternatively, the gel is incubated in deionized water. The gel is thenincubated in a solution that contains a colored or fluorescent dye thatselectively binds to the phosphoaffinity particles. The selected dyegenerally has little or no avidity for the proteins or gel matrix. Onefluorescent dye known to bind to silica in diatoms is2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole (PDMPO) and this compound can also interact withother metal oxides to serve as a fluorescent reporter (Shimizu et al,2001). Typically, sulfonated dyes are avoided as they are bind toproteins, though addition of a small molecule sulfonated compound canminimize this nonspecific interaction. The gel is then incubated againwith shaking and excess dye is removed. Phosphorylated polypeptides aredetected by visual observation, or by using a device such as a UV-basedCCD camera detection system, a laser-based gel scanner, axenon-arc-based CCD camera detection system, a Polaroid camera combinedwith a UV-transilluminator or a variety of other devices used fordetecting colored or fluorescent moieties. Similar approaches areemployed when detecting electrobloted phosphoproteins on polymericmembranes, such as nitrocellulose or polyvinylidene difluoridemembranes. Generally, incubation periods are shorter for transfermembranes due to better accessibility of polypeptides to detectionreagents.

EXAMPLE 11

This example describes detection of a phosphorylated polypeptide in amatrix using a pre-derivatized polypeptide sample.

Detection of phosphoproteins or phosphopeptides in gels or on blots canbe performed using a phosphoaffinity material with samplespre-derivatized with a fluorophore. In this case, the phosphoaffinitymaterial is a hydrated metal oxide such as yttrium oxide, yttriumaluminum garnet or titanium dioxide in particle or crystal form. Forlabeling of protein samples, succinimidyl esters of the cyanine dyes,Cy2, Cy3 and Cy5, can be employed to fluorescently label as many asthree different complex protein populations prior to mixing themtogether and running them simultaneously on the same 2D gel using amethod referred to as difference gel electrophoresis (DIGE) (Unlu et al,1997). Images of the 2D gels are acquired using as many as threedifferent excitation/emission filters, and the ratio of the differentlycolored fluorescent signals is used to find protein differences amongthe samples. DIGE allows two to three samples to be separated underidentical electrophoretic conditions, simplifying the process ofregistering and matching the gel images. One application of thistechnology is for examining differences between two samples (e.g.,drug-treated-vs-control cells or diseased-vs-healthy tissue). Gels orblots can be incubated in phosphoaffinity particles as described abovein Example 10. Certain particles can quench the signal from thefluorophore-labeled phosphoproteins and comparison of the fluorescentprofiles generated from gels before and after incubation with theparticles indicate which proteins in the profile are Phosphorylated bysubtractive analysis of the two images generated. A similar approach canbe used when the inherent fluorescence of proteins, arising primarilyfrom tryptophan fluorescence, is employed in order to visualize proteins(Roegener et al, 2003).

EXAMPLE 12

This example describes detecting phosphoaffinity materials orphosphomolecule-phosphoaffinity material complexes usinginductively-coupled plasma mass spectrometry.

This example describes one type of analytical approach for elementalanalysis—inductively-coupled plasma mass spectrometry (ICP-MS). UsingICP-MS, as little as 1 part per billion (ppb) of metal ions can bedetected. The ionized conversion of aluminum, which has a firstionization potential of 5.986 electron volts, is 99% under identical runconditions as described for phosphorous in Wilbur and McCurdy, 2001.Thus, following the procedure outlined in Wilbur and McCurdy, 2001 fordetecting Al (III) instead of phosphorous improves detection 16-fold. Inaddition, the specific detection of the metals can move the detectionwindow away from sample background signal.

For the sake of background information, inductively-coupled plasma massspectrometry (ICP-MS) is useful for trace elemental analysis ofenvironmental, biological, and pharmaceutical samples. Laser ablationICP-MS permits trace element analysis by combining the spatialresolution of an ultraviolet laser beam with the mass resolution andelement sensitivity of a modern ICP-MS. UV laser light, usually producedat a wavelength of 193-266 nm is focused on a sample surface, causingsample ablation. Ablation craters of 15-20 microns are routinelyproduced by the instrumentation. No special sample preparation isrequired for the procedure. Ablated material is transported in an argoncarrier gas directly to the high temperature inductively-coupled plasmaand the resulting ions are then drawn into a mass spectrometer fordetection and counting. A mass filter selects particles on the basis oftheir charge/mass ratio so that only specific isotopes are allowedthrough the filter and can enter the electron multiplier detectormounted at the end of the mass spectrometer (quadrupole, magnetic sectoror time-of-flight instrumentation). Detected signals of individualisotopes can be converted to isotopic ratios or, when standards aremeasured along with the unknowns, to the actual element concentrations.

ICP-MS-based detection of phosphoaffinity materials orphosphomolecule-phosphoaffinity material complexes can be performedaccording to the following exemplary procedure. First, phosphorylatedmolecules are bound to spots of immobilized binding partners (forexample, antibodies, aptamers, or any other affinity molecule selectivefor the molecules) contained on a microarray. Then, the array isincubated with one or more types of phosphoaffinity materials. Next, thearray is washed in a buffer, such as 50 mM sodium acetate, pH 6.0, 50 mMmagnesium chloride to remove excess phosphoaffinity material. Theindividual spots on the array are subjected to laser ablation ICP-MS bymethods similar to those described, for example, in Marshall et al, 2002and Wind et al, 2003, except that the relevant metal signal isquantified (rather than the phosphorous signal.) Detection using alaser-ablation ICP-mass spectrometer instrument is generally carried outby directing an ultraviolet laser ablation beam, usually in the form ofa collimated beam, toward a focusing lens. The lens can focus the beamto a high flux density on a particular microarray spot, causing localablation. The ablated molecules are captured in an ICP sampling tube,where they are carried by a flow of carrier gas away from themicroarray. The carrier gas is generally provided in a manner thatfloods the vicinity of the area subject to ablation. The carrier gas andablated molecules are carried to an ICP-mass spectrometer instrumentwhere the molecules are ionized in the plasma followed by massidentification in the spectrometer.

An exemplary ICP-MS-based detection procedure can involve the followingsteps. First, phosphomolecules are captured on a microarray containingantibodies, aptamers or other affinity molecules selective towardsmolecules of interest. Next, the array is incubated with a colloidalsolution of hydrated metal oxide particles. Next, the array is washedrepeatedly in a buffer, such as 50 mM sodium acetate, pH 6.0, 50 mMmagnesium chloride to remove excess colloidal metal particles. Theindividual spots on the array are subjected to laser ablation ICP-MS bymethods similar to those described in Marshall et al, 2002 and Wind etal, 2003, except that the relevant metal signal is quantified ratherthan the phosphorous signal. Sampling can be performed by single ormulti-spot analysis, straight line scans or rastering.

EXAMPLE 13

This example describes an exemplary solid support assay carried outusing a phosphoaffinity material incorporating a hydrated metal oxide,in particle form, as a detection agent.

The assay is carried out as follows. Binding partners for phosphorylatedmolecules are immobilized as spots on the surface of a solid support.The binding partners can be, for example, antibodies, peptides,aptamers, and the like. The spots can contain different binding partnersor can be replicate spots. A sample suspected to contain phosphorylatedmolecules capable of binding to the binding partners is applied to thesolid support surface, and the solid support is incubated. As withconventional microarray type support, incubation can be performed in theopen, under a coverslip, or in an incubation instrument. Afterincubation, the sample is washed from the solid support to remove excessand non-selectively bound phosphoaffinity particle and other molecules.Buffers containing inorganic phosphate, such as phosphate-bufferedsaline, are generally avoided because phosphate can compete for bindingof phosphoaffinity particles, and reduce the amount of phosphoaffinityparticles bound to phosphorylated molecules.

The spots now contain the binding partners plus whatever complementaryproteins have been bound to them during incubation with the sample. Asuspension of metal oxide nanoparticles, such as titanium dioxideparticles, is then applied to the solid support, allowed to incubate,and the excess is then is washed off. The phosphoaffinity particles bindselectively to the phosphorylated residues in proteins captured on thespots of the solid support.

At this stage, the spots include the binding partners plus the capturedcomplementary proteins with their phosphorylated residues labeled withmetal oxide nanoparticles. To enable fluorescent detection of thenanoparticles, a dye solution containing a metal oxide-specific dye,such as rhodamine B or rhodamine 6G, for example, is applied to thesolid support and incubated. After incubation, excess and unbound dye iswashed off.

Fluorescent detection of the dye is then performed using conventionalmethods. For example, a beam of excitation light comprising wavelengthsoverlapping the extinction band of the dye is directed to abeam-splitter element, which directs the excitation beam into theobjective lens. The lens focuses the beam onto a small area of the solidsupport surface. If dye is present at the focus, it emits fluorescencelight at a longer wavelength. A fraction of the emitted fluorescencelight is captured by the lens and formed into a beam. The beam-splitterpasses the fluorescence light to a detector, such as a photomultipliertube or CCD array. Typically an emission filter is placed before thedetector to block any wavelengths not generated by the dye fluorescence.The fluorescence signal is proportional to the local area concentrationof the dye, and hence to the local concentration of phosphorylatedresidues. In fluorescent detection, a variety of optical detectionarrangements can be used in place of the epi-fluorescent shown. Theexcitation can be evanescent or off-axis dark-field illumination, forexample. The emission can be imaged from a line or area of the substrateto a line or area array detector, or can be collected point-by-pointwith a scanning system and a single-element detector.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It can be understood that various modifications can be madewithout departing from the spirit of the invention.

The following publications are incorporated herein by reference, intheir entireties:

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1. A method for isolating a phosphomolecule from a sample, comprising:(a) contacting a sample with a phosphoaffinity material comprising ahydrated metal oxide, under conditions wherein a phosphomolecule iscapable of binding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, wherein the hydratedmetal oxide comprises yttrium, and (b) separating thephosphomolecule-phosphoaffinity material complex from the sample,thereby isolating the phosphomolecule from the sample.
 2. A method forisolating a phosphomolecule from a sample, comprising: (a) contacting asample with a phosphoaffinity material comprising a hydrated metal oxideand a support, under conditions wherein a phosphomolecule is capable ofbinding to the phosphoaffinity material to form aphosphomolecule-phosphoaffinity material complex, wherein the hydratedmetal oxide comprises yttrium, and (b) eluting a phosphomolecule fromthe phosphomolecule-phosphoaffinity material complex, thereby isolatingthe phosphomolecule from the sample.
 3. The method of claim 1 or 2,further comprising separating the phosphomolecule from thephosphomolecule-phosphoaffinity material complex.
 4. The method of claim2, further comprising removing unbound sample components from thephosphomolecule-phosphoaffinity material complex prior to eluting.
 5. Amethod for isolating a phosphorylated polypeptide from a sample,comprising: (a) contacting a sample with a phosphoaffinity materialcomprising a hydrated metal oxide and a support, under conditionswherein a phosphomolecule is capable of binding to the phosphoaffinitymaterial to form a phosphomolecule-phosphoaffinity material complex andin a liquid medium comprising an organic solvent, and (b) eluting aphosphomolecule from the phosphomolecule-phosphoaffinity materialcomplex, thereby isolating the phosphomolecule from the sample.
 6. Themethod of claim 5, wherein the liquid medium comprises isopropanol. 7.The method of claim 5, wherein the liquid medium comprises acetonitrile.8. The method of claim 5, wherein the eluting is performed in thepresence of a detergent.
 9. The method of claim 5, wherein the detergentis an ionic detergent.
 10. A method for detecting a phosphomolecule in asample, comprising: (a) contacting a sample with a phosphoaffinitymaterial comprising a hydrated metal oxide, under conditions wherein aphosphomolecule is capable of binding to the phosphoaffinity material toform a phosphomolecule-phosphoaffinity material complex, and (b)detecting formation of a phosphomolecule-phosphoaffinity materialcomplex, thereby detecting a phosphomolecule in the sample.
 11. Themethod of claim 10, wherein the detecting comprises measuring bindingbetween the phosphomolecule and phosphoaffinity material.
 12. The methodof claim 10, wherein the measuring comprises a mode selected from thegroup of absorbance, transmission, mass measurement, fluorescenceintensity, fluorescence polarization, fluorescence resonance energytransfer, time-resolved fluorescence, resonance light scattering,surface-enhanced Raman scattering, inductively-coupled plasma massspectrometry, electron paramagnetic resonance, refractive indexabsorbance, nuclear magnetic resonance, microcalorimetry, Fouriertransform infrared spectrometry, atomic spectrometry, surface plasmonresonance, refractive index changes, spectropolarimetry andellipsometry.
 13. The method of claim 10, wherein the detectingcomprises detecting the phosphoaffinity material portion of thephosphomolecule-phosphoaffinity material complex.
 14. The method ofclaim 10, wherein the metal of the phosphoaffinity material portion isdetected.
 15. The method of claim 10, wherein the metal is detectedusing a mass spectrometry mode.
 16. The method of claim 15, wherein themass spectrometry mode is inductively-coupled plasma mass spectrometry.17. The method of claim 10, further comprising labeling thephosphoaffinity material with a detectable tag prior to detecting. 18.The method of claim 17, wherein the detectable tag is a fluorescentmoiety.
 19. The method of claim 10, wherein the hydrated metal oxide islabeled with a fluorescent moiety.
 20. The method of claim 10, whereinthe detecting comprises detecting the phosphomolecule portion of thephosphomolecule-phosphoaffinity material complex.
 21. The method ofclaim 10, wherein the mass spectrometry mode is inductively-coupledplasma mass spectrometry.
 22. The method of claim 10, wherein thedetecting comprises detecting fluorescence resonance energy transfer.23. The method of claim 10, wherein fluorescence resonance energytransfer occurs between a fluorescent tag on the phosphomolecule and afluorescent tag on the phosphoaffinity material.
 24. The method of claim10, wherein the hydrated metal oxide is selected from the group ofaluminum oxide, titanium oxide, yttrium iron garnet, yttrium aluminumgarnet, yttrium gallium garnet, ferric oxide, gallium oxide, yttriumoxide, vanadium oxide, zirconium oxide, iron titanate, iron aluminate,calcium titanate, sodium titanate, zirconium aluminate, goethite,gibbsite, bayerite, boehmite, ilmenite, ilmenorutile, pseudorutile,rutile, brookite, pseudobrookite, geikielite, pyrophanite, ecandrewsite,melanostibite, armalcolite, srilankite and anatase.
 25. The method ofclaim 24, wherein the hydrated metal oxide is selected from the group ofyttrium oxide, yttrium iron garnet and titanium dioxide.
 26. The methodof any of claims 1, 2, 5 and 10, wherein the hydrated metal oxide isyttrium oxide.
 27. The method of any of claims 1, 2,5 and 10, whereinthe hydrated metal oxide is yttrium iron garnet.
 28. The method of anyof claims 1, 2, 5 and 10, wherein the hydrated metal oxide is inparticle form.
 29. The method of any of claims 1, 2, 5 and 10, whereinthe phosphoaffinity material further comprises a support.
 30. The methodof any of claims 1, 2, 5 and 10, wherein the support is selected fromthe group of particle, bead, gel, matrix, membrane, filter, fiber,sheet, mesh, frit, resin, sample vessel, column, pipette tip, slidechannel and MALDI-TOF plate.
 31. The method of any of claims 1, 2, 5 and10, wherein the support comprises an inorganic material.
 32. The methodof any of claims 1, 2, 5 and 10, wherein the support is a particle. 33.The method of claim 32, wherein the particle comprises a colloidalmetal.
 34. The method of any of claims 1, 2, 5 and 10, wherein thesupport comprises an organic material.
 35. The method of any of claims1, 2, 5 and 10, wherein the support is a sheet.
 36. The method of claim35, wherein the sheet comprises cellulose.
 37. The method of any ofclaims 1, 2, 5 and 10, wherein the support comprises a detectable tag.38. The method of any of claims 1, 2, 5 and 10, wherein thephosphomolecule is a macromolecule.
 39. The method of any of claims 1,2, 5 and 10, wherein the phosphomolecule is a phosphorylatedpolypeptide.
 40. The method of any of claims 1, 2, 5 and 10, wherein thesample comprises a support.
 41. The method of any of claims 1, 2, 5 and10, wherein the sample comprises a detectable tag.
 42. A commercialpackage, comprising a phosphoaffinity unit, the unit comprising aplurality of support sheets coated with a hydrated metal oxide.
 43. Thecommercial package of claim 42, wherein the hydrated metal oxide istitanium oxide.
 44. The commercial package of claim 43, wherein thesupport sheet is a membrane.
 45. The commercial package of claim 44,wherein the support sheet comprises cellulose.
 46. A commercial package,comprising a hydrated metal oxide attached to a support, wherein thehydrated metal oxide comprising yttrium.
 47. The commercial package ofclaim 46, wherein the hydrated metal oxide is yttrium oxide.
 48. Thecommercial package of claim 47, wherein the hydrated metal oxide isyttrium iron oxide.
 49. A method for isolating a phosphorylated moleculefrom a sample, as is set forth herein.
 50. A method for detecting aphosphorylated molecule from a sample, as is set forth herein.
 51. Acommercial package for performing a method for isolating or detecting aphosphorylated molecule, as is set forth herein.